ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites.

The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.

A Tour of TOR Complex Signaling in Plants.

To identify the appropriate times for growth and development, organisms must sense and process information about the availability of nutrients, energy status, and environmental cues. For sessile eukaryotes such as plants, integrating such information can be critical in life or death decisions. For nearly 30 years, the conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs) target of rapamycin (TOR) has been established as a central hub for integrating external and internal metabolic cues. Despite the functional conservation across eukaryotes, the TOR complex has evolved specific functional and mechanistic features in plants. Here, we present recent findings on the plant TOR complex that highlight the conserved and unique nature of this critical growth regulator and its role in multiple aspects of plant life.

Microtubule bundling by MAP65-1 protects against severing by inhibiting the binding of katanin.

The microtubule-severing enzyme katanin (KTN1) regulates the organization and turnover of microtubule arrays by the localized breakdown of microtubule polymers. In land plants, KTN1 activity is essential for the formation of linearly organized cortical microtubule arrays that determine the axis of cell expansion. Cell biological studies have shown that even though KTN1 binds to the sidewalls of single and bundled microtubules, severing activity is restricted to microtubule cross-over and nucleation sites, indicating that cells contain protective mechanisms to prevent indiscriminate microtubule severing. Here, we show that the microtubule-bundling protein MAP65-1 inhibits KTN1-mediated microtubule severing in vitro. Severing is inhibited at bundled microtubule segments and the severing rate of nonbundled microtubules is reduced by MAP65-1 in a concentration-dependent manner. Using various MAP65-1 mutant proteins, we demonstrate that efficient cross-linking of microtubules is crucial for this protective effect and that microtubule binding alone is not sufficient. Reduced severing due to microtubule bundling by MAP65-1 correlated to decreased binding of KTN1 to these microtubules. Taken together, our work reveals that cross-linking of microtubules by MAP65-1 confers resistance to severing by inhibiting the binding of KTN1 and identifies the structural features of MAP65-1 that are important for this activity.

Systematic survey of the function of ROP regulators and effectors during tip growth in the moss Physcomitrella patens.

Rho/Rac of plants (ROP) GTPases are plant-specific small GTPases that regulate cell morphology. ROP activity is controlled by several families of regulatory proteins. However, how these diverse regulators contribute to polarized growth remains understudied. In a system-wide approach, we used RNAi to silence each gene family of known ROP regulators in the juvenile tissues of the moss Physcomitrella patens. We found that the GTPase activating proteins, but not the ROP enhancers, are essential for tip growth. The guanine exchange factors (GEFs), which are comprised of ROPGEFs and Spikes, both contribute to growth. However, silencing Spikes results in less-polarized plants as compared to silencing ROPGEFs, suggesting that Spikes contribute more to establishing cell polarity. Silencing the single-gene family of guanine dissociation inhibitors also inhibits growth, resulting in small, unpolarized plants. In contrast, silencing the ROP effector ROP-interactive CRIB-containing (RIC) protein, which is encoded by a single gene, results in plants larger than the controls, suggesting that RIC functions to inhibit tip growth in moss. Taken together, this systematic loss-of-function survey provides insights into the function of ROP regulators during polarized growth.

The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus Ends.

The contribution of microtubule tip dynamics to the assembly and function of plant microtubule arrays remains poorly understood. Here, we report that the Arabidopsis SPIRAL2 (SPR2) protein modulates the dynamics of the acentrosomal cortical microtubule plus and minus ends in an opposing manner. Live imaging of a functional SPR2-mRuby fusion protein revealed that SPR2 shows both microtubule plus- and minus-end tracking activity in addition to localization at microtubule intersections and along the lattice. Analysis of microtubule dynamics showed that cortical microtubule plus ends rarely undergo catastrophe in the spr2-2 knockout mutant compared to wild-type. In contrast, cortical microtubule minus ends in spr2-2 depolymerized at a much faster rate than in wild-type. Destabilization of the minus ends in spr2-2 caused a significant decrease in the lifetime of microtubule crossovers, which dramatically reduced the microtubule-severing frequency and inhibited light-induced microtubule array reorientation. Using in vitro reconstitution experiments combined with single-molecule imaging, we found that recombinant SPR2-GFP intrinsically localizes to microtubule minus ends, where it binds stably and inhibits their dynamics. Together, our data establish SPR2 as a new type of microtubule tip regulator that governs the length and lifetime of microtubules.

A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion.

In plants, the ROP family of small GTPases has been implicated in the polarized growth of tip-growing cells, such as root hairs and pollen tubes; however, most of the data derive from overexpressing ROP genes or constitutively active and dominant-negative isoforms, whereas confirmation by using loss-of-function studies has generally been lacking. Here, in the model moss Physcomitrella patens, we study ROP signaling during tip growth by using a loss-of-function approach based on RNA interference (RNAi) to silence the entire moss ROP family. We find that plants with reduced expression of ROP genes, in addition to failing to initiate tip growth, have perturbed cell wall staining, reduced cell adhesion and have increased actin-filament dynamics. Although plants subjected to RNAi against the ROP family also have reduced microtubule dynamics, this reduction is not specific to loss of ROP genes, as it occurs when actin function is compromised chemically or genetically. Our data suggest that ROP proteins polarize the actin cytoskeleton by suppressing actin-filament dynamics, leading to an increase in actin filaments at the site of polarized secretion.

Myosin XI is essential for tip growth in Physcomitrella patens.

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5' untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.

A putative transporter is essential for integrating nutrient and hormone signaling with lateral root growth and nodule development in Medicago truncatula.

Legume root architecture involves not only elaboration of the root system by the formation of lateral roots but also the formation of symbiotic root nodules in association with nitrogen-fixing soil rhizobia. The Medicago truncatula LATD/NIP gene plays an essential role in the development of both primary and lateral roots as well as nodule development. We have cloned the LATD/NIP gene and show that it encodes a member of the NRT1(PTR) transporter family. LATD/NIP is expressed throughout the plant. pLATD/NIP-GFP promoter-reporter fusions in transgenic roots establish the spatial expression of LATD/NIP in primary root, lateral root and nodule meristems and the surrounding cells. Expression of LATD/NIP is regulated by hormones, in particular by abscisic acid which has been previously shown to rescue the primary and lateral root meristem arrest of latd mutants. latd mutants respond normally to ammonium but have defects in responses of the root architecture to nitrate. Taken together, these results suggest that LATD/NIP may encode a nitrate transporter or transporter of another compound.

Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.

Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth.