The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions ...caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.
Abbreviations: ATG: autophagy related; CLP: caspase-like protease; HR: hypersensitive response; PCD: programmed cell death; St: Solanum tuberosum; VPE: vacuolar processing enzyme.
Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement ...in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.
Plant organelles are highly motile, with speed values of 3-71μm/s in cells of land plants and about 20-60 μm/s in characean algal cells. This movement is believed to be important for rapid ...distribution of materials around the cell, for the plant＇s ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins.Most plants express multiple members of two main classes： myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate.
Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional ...myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB.
In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding.
Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity.
It has recently been found that among the 17 Arabidopsis myosins, six (XIC, XIE, XIK, XI-I, MYA1, and MYA2) have a major role in the motility of Golgi bodies and mitochondria in Nicotiana benthamiana ...and Nicotiana tabacum. Here, the same dominant negative tail fragments were also found to arrest the movement of Gogi bodies when transiently expressed in Arabidopsis plants. However, when a Golgi marker was transiently expressed in plants knocked out in these myosins, its movement was dramatically inhibited only in the xik mutant. In addition, a tail fragment of myosin XIK could inhibit the movement of several post-Golgi organelles, such as the trans-Golgi network, pre-vacuolar compartment, and endosomes, as well as total cytoplasmic streaming, suggesting that myosin XIK is a major player in cytoplasm kinetics. However, no co-localization of myosin tails with the arrested organelles was observed. Several deletion truncations of the myosin XIK tail were generated to corroborate function with localization. All deletion mutants possessing an inhibitory effect on organelle movement exhibited a diffuse cytoplasmic distribution. Point mutations in the tail of myosin XIK revealed that Arg1368 and Arg1443 are essential for its activity. These residues correspond to Lys1706 and Lys1779 from mouse myosin Va, which mediate the inhibitory head–tail interaction in this myosin. Therefore, such an interaction might underlie the dominant negative effect of truncated plant myosin tails and explain the mislocalization with target organelles.
How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates ...developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6
DN
) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6
DN
plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6 DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 npr1) or biosynthesis (salicylic acid induction deficient2 sid2). However, the rop6
DN
npr1 and rop6
DN
sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6
DN
plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6
DN
plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6
DN
plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution.
The distribution of myosin VIII ATM1 tail in association with the plasma membrane is often observed in coordination with that of cortical microtubules (MTs). The prevailing hypothesis is that ...coordination between the organization of cortical MTs and proteins in the membrane results from the inhibition of free lateral diffusion of the proteins by barriers formed by MTs. Since the positioning of myosin VIII tail in the membrane is relatively stable, we ask: can it affect the organization of MTs? Myosin VIII ATM1 tail co-localized with remorin 6.6, the position of which in the plasma membrane is also relatively stable. Overexpression of myosin VIII ATM1 tail led to a larger fraction of MTs with a lower rate of orientation dispersion. In addition, collisions between MTs and cortical structures labeled by ATM1 tail or remorin 6.6 were observed. Collisions between EB1 labeled MTs and ATM1 tail clusters led to four possible outcomes: 1-Passage of MTs through the cluster; 2-Decreased elongation rate; 3-Disengagement from the membrane followed by a change in direction; and 4-retraction. EB1 tracks became straighter in the presence of ATM1 tail. Taken together, collisions of MTs with ATM1 tail labeled structures can contribute to their coordinated organization.
Argania spinosa
trees have attracted attention in recent years due to their high resistance to extreme climate conditions. Initial domestication activities practiced in Morocco. Here we report on ...selection and vegetative propagation of
A. spinosa
trees grown in Israel. Trees yielding relatively high amounts of fruit were propagated by rooting of stem cuttings. High variability in rooting ability was found among the 30 clones selected. In-depth comparison of a difficult-to-root (ARS7) and easy-to-root (ARS1) clone revealed that the rooted cuttings of ARS7 have a lower survival rate than those of ARS1. In addition, histological analysis of the adventitious root primordia showed many abnormal fused primordia in ARS7. Hormone profiling revealed that while ARS1 accumulates more cytokinin, ARS7 accumulates more auxin, suggesting different auxin-to-cytokinin ratios underlying the different rooting capabilities. The hypothesized relationship between rooting and grafting abilities was addressed. Reciprocal grafting was performed with ARS1/ARS7 but no significant differences in the success of graft unification between the trees was detected. Accordingly, comparative RNA sequencing of the rooting and grafting zones showed more differentially expressed genes related to rooting than to grafting between the two trees. Clustering, KEGG and Venn analyses confirmed enrichment of genes related to auxin metabolism, transport and signaling, cytokinin metabolism and signaling, cell wall modification and cell division in both regions. In addition, the differential expression of some key genes in ARS1 vs. ARS7 rooting zones was revealed. Taken together, while both adventitious root-formation and graft-unification processes share response to wounding, cell reprogramming, cell division, cell differentiation and reconnection of the vasculature, there are similar, but also many different genes regulating the two processes. Therefore an individual genotype can have low rooting capacity but good graft-unification ability.
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