The TOR complex controls ATP levels to regulate actin cytoskeleton dynamics in Arabidopsis.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B, or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.

A G protein-coupled receptor-like module regulates cellulose synthase secretion from the endomembrane system in Arabidopsis.

Cellulose is produced at the plasma membrane of plant cells by cellulose synthase (CESA) complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked to the plasma membrane. Because CESAs are only active in the plasma membrane, control of CSC secretion regulates cellulose synthesis. We identified members of a family of seven transmembrane domain-containing proteins (7TMs) that are important for cellulose production during cell wall integrity stress. 7TMs are often associated with guanine nucleotide-binding (G) protein signaling and we found that mutants affecting the Gßγ dimer phenocopied the 7tm mutants. Unexpectedly, the 7TMs localized to the Golgi/trans-Golgi network where they interacted with G protein components. Here, the 7TMs and Gßγ regulated CESA trafficking but did not affect general protein secretion. Our results outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses to cell wall integrity stress.

Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

Plant science's next top models.

BACKGROUND: Model organisms are at the core of life science research. Notable examples include the mouse as a model for humans, baker's yeast for eukaryotic unicellular life and simple genetics, or the enterobacteria phage λ in virology. Plant research was an exception to this rule, with researchers relying on a variety of non-model plants until the eventual adoption of Arabidopsis thaliana as primary plant model in the 1980s. This proved to be an unprecedented success, and several secondary plant models have since been established. Currently, we are experiencing another wave of expansion in the set of plant models. SCOPE: Since the 2000s, new model plants have been established to study numerous aspects of plant biology, such as the evolution of land plants, grasses, invasive and parasitic plant life, adaptation to environmental challenges, and the development of morphological diversity. Concurrent with the establishment of new plant models, the advent of the 'omics' era in biology has led to a resurgence of the more complex non-model plants. With this review, we introduce some of the new and fascinating plant models, outline why they are interesting subjects to study, the questions they will help to answer, and the molecular tools that have been established and are available to researchers. CONCLUSIONS: Understanding the molecular mechanisms underlying all aspects of plant biology can only be achieved with the adoption of a comprehensive set of models, each of which allows the assessment of at least one aspect of plant life. The model plants described here represent a step forward towards our goal to explore and comprehend the diversity of plant form and function. Still, several questions remain unanswered, but the constant development of novel technologies in molecular biology and bioinformatics is already paving the way for the next generation of plant models.

Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis.

Asparagus Spears as a Model to Study Heteroxylan Biosynthesis during Secondary Wall Development.

Garden asparagus (Asparagus officinalis L.) is a commercially important crop species utilized for its excellent source of vitamins, minerals and dietary fiber. However, after harvest the tissue hardens and its quality rapidly deteriorates because spear cell walls become rigidified due to lignification and substantial increases in heteroxylan content. This latter observation prompted us to investigate the in vitro xylan xylosyltransferase (XylT) activity in asparagus. The current model system for studying heteroxylan biosynthesis, Arabidopsis, whilst a powerful genetic system, displays relatively low xylan XylT activity in in vitro microsomal preparations compared with garden asparagus therefore hampering our ability to study the molecular mechanism(s) of heteroxylan assembly. Here, we analyzed physiological and biochemical changes of garden asparagus spears stored at 4 °C after harvest and detected a high level of xylan XylT activity that accounts for this increased heteroxylan. The xylan XylT catalytic activity is at least thirteen-fold higher than that reported for previously published species, including Arabidopsis and grasses. A biochemical assay was optimized and up to seven successive Xyl residues were incorporated to extend the xylotetraose (Xyl4) acceptor backbone. To further elucidate the xylan biosynthesis mechanism, we used RNA-seq to generate an Asparagus reference transcriptome and identified five putative xylan biosynthetic genes (AoIRX9, AoIRX9-L, AoIRX10, AoIRX14_A, AoIRX14_B) with AoIRX9 having an expression profile that is distinct from the other genes. We propose that Asparagus provides an ideal biochemical system to investigate the biochemical aspects of heteroxylan biosynthesis and also offers the additional benefit of being able to study the lignification process during plant stem maturation.

Isolation and functional analysis of CONSTANS-LIKE genes suggests that a central role for CONSTANS in flowering time control is not evolutionarily conserved in Medicago truncatula.

The zinc finger transcription factor CONSTANS has a well-established central role in the mechanism for photoperiod sensing in Arabidopsis, integrating light and circadian clock signals to upregulate the florigen gene FT under long-day but not short-day conditions. Although CONSTANS-LIKE (COL) genes in other species have also been shown to regulate flowering time, it is not clear how widely this central role in photoperiod sensing is conserved. Legumes are a major plant group and various legume species show significant natural variation for photoperiod responsive flowering. Orthologs of several Arabidopsis genes have been shown to participate in photoperiodic flowering in legumes, but the possible function of COL genes as integrators of the photoperiod response has not yet been examined in detail. Here we characterize the COL family in the temperate long-day legume Medicago truncatula, using expression analyses, reverse genetics, transient activation assays and Arabidopsis transformation. Our results provide several lines of evidence suggesting that COL genes are unlikely to have a central role in the photoperiod response mechanism in this species.

Transient gene expression in Medicago truncatula leaves via Agroinfiltration.

Transient expression is a powerful method for the functional characterization of genes. In this chapter, we outline a protocol for the transient expression of constructs in Medicago truncatula leaves using Agrobacterium tumefaciens infiltration. Using quantitative real-time PCR we demonstrate that the infiltration of a construct containing the LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1) transcription factor results in the strong upregulation of key biosynthetic genes and the accumulation of anthocyanin pigment in the leaves after just 3 days. Thus, this method provides a rapid and powerful way to the discovery of downstream targets of M. truncatula transcription factors.