A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

Cell Death in Cells Overlying Lateral Root Primordia Facilitates Organ Growth in Arabidopsis.

Plant organ growth is widely accepted to be determined by cell division and cell expansion, but, unlike that in animals, the contribution of cell elimination has rarely been recognized. We investigated this paradigm during Arabidopsis lateral root formation, when the lateral root primordia (LRP) must traverse three overlying cell layers within the parent root. A subset of LRP-overlying cells displayed the induction of marker genes for cell types undergoing developmental cell death, and their cell death was detected by electron, confocal, and light sheet microscopy techniques. LRP growth was delayed in cell-death-deficient mutants lacking the positive cell death regulator ORESARA1/ANAC092 (ORE1). LRP growth was restored in ore1-2 knockout plants by genetically inducing cell elimination in cells overlying the LRP or by physically killing LRP-overlying cells by ablation with optical tweezers. Our results support that, in addition to previously discovered mechanisms, cell elimination contributes to regulating lateral root emergence.

Precision Automation of Cell Type Classification and Sub-Cellular Fluorescence Quantification from Laser Scanning Confocal Images.

While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to (1) segment radial plant organs into individual cells, (2) classify cells into cell type categories based upon Random Forest classification, (3) divide each cell into sub-regions, and (4) quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types.

Vibrational spectroscopic image analysis of biological material using multivariate curve resolution-alternating least squares (MCR-ALS).

Raman and Fourier transform IR (FTIR) microspectroscopic images of biological material (tissue sections) contain detailed information about their chemical composition. The challenge lies in identifying changes in chemical composition, as well as locating and assigning these changes to different conditions (pathology, anatomy, environmental or genetic factors). Multivariate data analysis techniques are ideal for decrypting such information from the data. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and unmixing of pixel spectra into their contributing pure components by multivariate curve resolution-alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile in order to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually <30-60 min per image), and they are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or heat maps of single-band intensities. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k-means clustering) is possible.

Class I KNOX transcription factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl.

The class I KNOX transcription factors SHOOT MERISTEMLESS (STM) and KNAT1 are important regulators of meristem maintenance in shoot apices, with a dual role of promoting cell proliferation and inhibiting differentiation. We examined whether they control stem cell maintenance in the cambium of Arabidopsis hypocotyls, a wood-forming lateral meristem, in a similar fashion as in the shoot apical meristem. Weak loss-of-function alleles of KNAT1 and STM led to reduced formation of xylem fibers - highly differentiated cambial derivatives - whereas cell proliferation in the cambium was only mildly affected. In a knat1;stm double mutant, xylem fiber differentiation was completely abolished, but residual cambial activity was maintained. Expression of early and late markers of xylary cell differentiation was globally reduced in the knat1;stm double mutant. KNAT1 and STM were found to act through transcriptional repression of the meristem boundary genes BLADE-ON-PETIOLE 1 (BOP1) and BOP2 on xylem fiber differentiation. Together, these data indicate that, in the cambium, KNAT1 and STM, contrary to their function in the shoot apical meristem, promote cell differentiation through repression of BOP genes.

Immunoprofiling reveals unique cell-specific patterns of wall epitopes in the expanding Arabidopsis stem.

The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

Transcriptional programming during cell wall maturation in the expanding Arabidopsis stem.

BACKGROUND: Plant cell walls are complex dynamic structures that play a vital role in coordinating the directional growth of plant tissues. The rapid elongation of the inflorescence stem in the model plant Arabidopsis thaliana is accompanied by radical changes in cell wall structure and chemistry, but analysis of the underlying mechanisms and identification of the genes that are involved has been hampered by difficulties in accurately sampling discrete developmental states along the developing stem. RESULTS: By creating stem growth kinematic profiles for individual expanding Arabidopsis stems we have been able to harvest and pool developmentally-matched tissue samples, and to use these for comparative analysis of global transcript profiles at four distinct phases of stem growth: the period of elongation rate increase, the point of maximum growth rate, the point of stem growth cessation and the fully matured stem. The resulting profiles identify numerous genes whose expression is affected as the stem tissues pass through these defined growth transitions, including both novel loci and genes identified in earlier studies. Of particular note is the preponderance of highly active genes associated with secondary cell wall deposition in the region of stem growth cessation, and of genes associated with defence and stress responses in the fully mature stem. CONCLUSIONS: The use of growth kinematic profiling to create tissue samples that are accurately positioned along the expansion growth continuum of Arabidopsis inflorescence stems establishes a new standard for transcript profiling analyses of such tissues. The resulting expression profiles identify a substantial number of genes whose expression is correlated for the first time with rapid cell wall extension and subsequent fortification, and thus provide an important new resource for plant biologists interested in gene discovery related to plant biomass accumulation.

Identification of quantitative trait loci controlling fibre length and lignin content in Arabidopsis thaliana stems.

Fibre properties and the biochemical composition of cell walls are important traits in many applications. For example, the lengths of fibres define the strength and quality of paper, and lignin content is a critical parameter for the use of biomass in biofuel production. Identifying genes controlling these traits is comparatively difficult in woody species, because of long generation times and limited amenability to high-resolution genetic mapping. To address this problem, this study mapped quantitative trait loci (QTLs) defining fibre length and lignin content in the Arabidopsis recombinant inbred line population Col-4 × Ler-0. Adapting high-throughput phenotyping techniques for both traits for measurements in Arabidopsis inflorescence stems identified significant QTLs for fibre length on chromosomes 2 and 5, as well as one significant QTL affecting lignin content on chromosome 2. For fibre length, total variation within the population was 208% higher than between parental lines and the identified QTLs explained 50.58% of the observed variation. For lignin content, the values were 261 and 26.51%, respectively. Bioinformatics analysis of the associated intervals identified a number of candidate genes for fibre length and lignin content. This study demonstrates that molecular mapping of QTLs pertaining to wood and fibre properties is possible in Arabidopsis, which substantially broadens the use of Arabidopsis as a model species for the functional characterization of plant genes.

Developmentally equivalent tissue sampling based on growth kinematic profiling of Arabidopsis inflorescence stems.

• Directional growth in Arabidopsis thaliana during bolting of the inflorescence stem makes this an attractive system for study of the underlying processes of tissue elongation and cell wall extension. Analysis of local molecular events accompanying Arabidopsis inflorescence stem elongation is hampered by difficulties in isolating developmentally matched tissue samples from different plants. • Here, we present a novel sampling approach in which specific developmental stages along the developing stem are defined nonintrusively in terms of their relative elemental growth rate by use of time-lapse imagery and subsequent derivation of growth kinematic profiles for individual plants. • Growth kinematic profiling reveals that key developmental transitions such as the point of maximum elongation rate and the point of cessation of elongation occur over broad and overlapping ranges across individuals within a population of the Columbia (Col-0) ecotype. The position of these transitions is only weakly correlated with overall plant height, which undermines the common assumption that physically similar plants have closely matched growth profiles. • This kinematic profiling approach provides high-resolution growth phenotyping of the developing stem and thereby enables the harvest, pooling and analysis of developmentally matched tissue samples from multiple Arabidopsis plants.

Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid.

Earlier studies have shown that RACK1 functions as a negative regulator of abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana), but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, coexpression analysis revealed that more than 80% of the genes coexpressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1's function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and Saccharomyces cerevisiae RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B, and RACK1C, complemented the growth defects of the S. cerevisiae cross pathway control2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor6 (eIF6), whose mammalian homolog is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

MYB75 functions in regulation of secondary cell wall formation in the Arabidopsis inflorescence stem.

Deposition of lignified secondary cell walls in plants involves a major commitment of carbon skeletons in both the form of polysaccharides and phenylpropanoid constituents. This process is spatially and temporally regulated by transcription factors, including a number of MYB family transcription factors. MYB75, also called PRODUCTION OF ANTHOCYANIN PIGMENT1, is a known regulator of the anthocyanin branch of the phenylpropanoid pathway in Arabidopsis (Arabidopsis thaliana), but how this regulation might impact other aspects of carbon metabolism is unclear. We established that a loss-of-function mutation in MYB75 (myb75-1) results in increased cell wall thickness in xylary and interfascicular fibers within the inflorescence stem. The total lignin content and S/G ratio of the lignin monomers were also affected. Transcript profiles from the myb75-1 inflorescence stem revealed marked up-regulation in the expression of a suite of genes associated with lignin biosynthesis and cellulose deposition, as well as cell wall modifying proteins and genes involved in photosynthesis and carbon assimilation. These patterns suggest that MYB75 acts as a repressor of the lignin branch of the phenylpropanoid pathway. Since MYB75 physically interacts with another secondary cell wall regulator, the KNOX transcription factor KNAT7, these regulatory proteins may form functional complexes that contribute to the regulation of secondary cell wall deposition in the Arabidopsis inflorescence stem and that integrate the metabolic flux through the lignin, flavonoid, and polysaccharide pathways.

SIPK conditions transcriptional responses unique to either bacterial or oomycete elicitation in tobacco.

SUMMARY The mitogen-activated protein kinase, SIPK (salicylic acid-induced protein kinase), is known to be rapidly activated in tobacco (Nicotiana tabacum) by various elicitors. However, SIPK activation induced by the oomycete elicitor, beta-megaspermin, is reported to require external calcium influx, whereas that induced by the bacterial elicitor, hrpZ(Psph), does not. This suggests that SIPK activation is involved in different elicitor-initiated signalling pathways, and raises the question of whether the role(s) of SIPK in mediating stress outcomes, including transcriptional re-programming, differs in an elicitor-specific manner. To examine this, we compared the impact of silencing SIPK on the transcript profile of tobacco suspension culture cells challenged with either hrpZ(Psph) or beta-megaspermin. SIPK-silencing was found to have a substantial impact on both hrpZ(Psph)- and beta-megaspermin-induced transcriptional responses, and these impacts included both common and elicitor-differentiated features. As well as revealing a role for SIPK in modulating expression of known redox- and defence-related genes in response to both elicitors, our analysis detected a substantial impact of SIPK silencing on transcription of 80S ribosomal subunit mRNAs. This novel observation suggests that SIPK may play a role in affecting translation efficiency as one mechanism for enacting rapid genome-wide, elicitor-specific physiological reprogramming during defence responses.

SIPK signaling controls multiple components of harpin-induced cell death in tobacco.

Harpin from Pseudomonas syringae pv. phaseolicola (HrpZ) elicits a rapid cell death response in tobacco plants. Multiple signaling components, including mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS) and salicylic acid (SA), have been reported to be involved in this cell death process, but the interaction between these molecules is poorly understood. Here we show through utilizing plants manipulated in SIPK expression levels that lack of SIPK results in increased sensitivity to harpin with concomitant accumulation of higher levels of ROS. Conversely, SIPK-overexpressing plants show reduced sensitivity to harpin relative to wild-type plants, and display reduced ROS accumulation. Harpin-induced cell death was found to be conditional on the ability of the plant to accumulate SA, whereas harpin induction of MAPK activation and ROS accumulation are not. However, harpin-induced ROS accumulation is required for activation of SIPK and wound-induced protein kinase. Transcriptional profiling revealed that suppression of SIPK signaling also affects early expression of a range of pathogen- and stress-responsive genes during harpin challenge.