Regulation of expression of calmodulin and calmodulin-related genes by environmental stimuli in plants.

Plants are very sensitive to environmental stimuli and have evolved the ability to adapt to many environmental stresses by altering development. In particular, mechanical stimuli such as touch or wind, result in growth changes that result in plants with greater resistance to such mechanical stimuli. We have initiated a molecular dissection of the pathways that enable perception of and responses to these environmental stimuli in plants. We have discovered five genes--termed the TCH genes--whose expression levels are strongly and rapidly increased in response to stimuli such as touch, wind, rain, wounding and darkness. Three of the TCH genes encode proteins related to calmodulin thereby implicating roles for calcium ions and calmodulin in the transduction of signals from the environment.     

Life in a changing world: TCH gene regulation of expression and responses to environmental signals

The Arabidopsis TCH genes were discovered as a consequence of their marked upregulation of expression in response to seemingly innocuous stimuli, such as touch. Further analyses have indicated that these genes are upregulated by a variety of diverse stimuli. Understanding the mechanism(s) and factors that control TCH gene regulation will shed light on the signalling pathways that enable plants to respond to changing environmental conditions. The TCH proteins include calmodulin, calmodulin-related proteins and a xyloglucan endotransglycosylase. Expression analyses and localization of protein accumulation indicate that the potential sites of TCH protein function include expanding cells and tissues under mechanical strain. We hypothesize that the TCH proteins may collaborate in cell wall biogenesis.     

Ionic signaling in plant responses to gravity and touch

Touch and gravity are two of the many stimuli that plants must integrate to generate an appropriate growth response. Due to the mechanical nature of both of these signals, shared signal transduction elements could well form the basis of the cross-talk between these two sensory systems. However, touch stimulation must elicit signaling events across the plasma membrane whereas gravity sensing is thought to represent transformation of an internal force, amyloplast sedimentation, to signal transduction events. In addition, factors such as turgor pressure and presence of the cell wall may also place unique constraints on these plant mechanosensory systems. Even so, the candidate signal transduction elements in both plant touch and gravity sensing, changes in Ca2+, pH and membrane potential, do mirror the known ionic basis of signaling in animal mechanosensory cells. Distinct spatial and temporal signatures of Ca2+ ions may encode information about the different mechanosignaling stimuli. Signals such as Ca2+ waves or action potentials may also rapidly transfer information perceived in one cell throughout a tissue or organ leading to the systemic reactions characteristic of plant touch and gravity responses. Longer-term growth responses are likely sustained via changes in gene expression and asymmetries in compounds such as inositol-1,4,5-triphosphate (IP3) and calmodulin. Thus, it seems likely that plant mechanoperception involves both spatial and temporal encoding of information at all levels, from the cell to the whole plant. Defining this patterning will be a critical step towards understanding how plants integrate information from multiple mechanical stimuli to an appropriate growth response.     

Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.     

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase.

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.     

Wind-induced mechanical stimulation increases pest resistance in common bean

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. Induction of enzymes such as phenylalanine ammonia-lyase and peroxidase and the accumulation of such phenolics as lignin can occur in response to insect and pathogen attack, exposure to oxidizing pollutants, and mechanical stimulation, and are thought to function in the resistance of plants to damage by these stresses. I investigated whether induction of components of this generalized stress response by wind-induced mechanical stimulation could influence the resistance to pests of common bean. In greenhouse studies, exposure of 7- to 10-day-old bean seedlings to daily periods of fan-produced wind led to increased activities of peroxidase and cinnamyl alcohol-dehydrogenase and enhanced the accumulation of lignin in primary leaves of these plants. Egg production and population growth of two-spotted spider mites were reduced when offered leaves of mechanically-stimulated plants in leaf-disk and whole-plant bioassays. Infection by anthracnose after inoculation in a detached-leaf bioassay was also reduced in leaves of mechanically-stimulated plants. The consistent positive association between the enhanced activity of the lignin branch of the phenylpropanoid pathway and enhanced resistance to pests found in leaves of mechanically-stimulated plants illustrates one way in which exposure of plants to environmental stimuli that activate a generalized stress response (e.g., wind) can influence the interactions of those plants with other environmental stimuli (e.g., pests). Received: 1 October 1996 / Accepted: 23 January 1997     

The regulation of cell wall extensibility during shade avoidance: a study using two contrasting ecotypes of Stellaria longipes

Shade avoidance in plants involves rapid shoot elongation to grow toward the light. Cell wall-modifying mechanisms are vital regulatory points for control of these elongation responses. Two protein families involved in cell wall modification are expansins and xyloglucan endotransglucosylase/hydrolases. We used an alpine and a prairie ecotype of Stellaria longipes differing in their response to shade to study the regulation of cell wall extensibility in response to low red to far-red ratio (R/FR), an early neighbor detection signal, and dense canopy shade (green shade: low R/FR, blue, and total light intensity). Alpine plants were nonresponsive to low R/FR, while prairie plants elongated rapidly. These responses reflect adaptation to the dense vegetation of the prairie habitat, unlike the alpine plants, which almost never encounter shade. Under green shade, both ecotypes rapidly elongate, showing that alpine plants can react only to a deep shade treatment. Xyloglucan endotransglucosylase/hydrolase activity was strongly regulated by green shade and low blue light conditions but not by low R/FR. Expansin activity, expressed as acid-induced extension, correlated with growth responses to all light changes. Expansin genes cloned from the internodes of the two ecotypes showed differential regulation in response to the light manipulations. This regulation was ecotype and light signal specific and correlated with the growth responses. Our results imply that elongation responses to shade require the regulation of cell wall extensibility via the control of expansin gene expression. Ecotypic differences demonstrate how responses to environmental stimuli are differently regulated to survive a particular habitat.     

Arabidopsis touch-induced morphogenesis is jasmonate mediated and protects against pests

Plants cannot change location to escape stressful environments. Therefore, plants evolved to respond and acclimate to diverse stimuli, including the seemingly innocuous touch stimulus [1-4]. Although some species, such as Venus flytrap, have fast touch responses, most plants display more gradual touch-induced morphological alterations, called thigmomorphogenesis [2, 3, 5, 6]. Thigmomorphogenesis may be adaptive; trees subjected to winds develop less elongated and thicker trunks and thus are less likely damaged by powerful wind gusts [7]. Despite the widespread relevance of thigmomorphogenesis, the regulation that underlies plant mechanostimulus-induced morphological responses remains largely unknown. Furthermore, whether thigmomorphogenesis confers additional advantage is not fully understood. Although aspects of thigmomorphogenesis resemble ethylene effects [8], and touch can induce ethylene synthesis [9, 10], Arabidopsis ethylene response mutants show touch-induced thigmomorphogenesis [11]; thus, ethylene response is nonessential for thigmomorphogenesis. Here we show that jasmonate (JA) phytohormone both is required for and promotes the salient characteristics of thigmomorphogenesis in Arabidopsis, including a touch-induced delay in flowering and rosette diameter reduction. Furthermore, we find that repetitive mechanostimulation enhances Arabidopsis pest resistance in a JA-dependent manner. These results highlight an important role for JA in mediating mechanostimulus-induced plant developmental responses and resultant cross-protection against biotic stress.     

In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation

In the nematode C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indicator cameleon to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. We defined a gentle touch sensory modality that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation.     

Alfalfa Cellulose Synthase Gene Expression under Abiotic Stress: A Hitchhiker’s Guide to RT-qPCR Normalization

Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L.), no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress) at various time points (e.g. 0, 24, 72 and 96 h). We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots), under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.     

Mechanotransduction in Caenorhabditis elegans

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or mechanical force delivered to a cell, are interpreted to direct biological responses. All living organisms, and probably all cells, have the ability to sense and respond to mechanical stimuli. At the single-cell level, mechanical signaling underlies cell-volume control and specialized responses such as the prevention of poly-spermy in fertilization. At the level of the whole organism, mechanotransduction underlies processes as diverse as stretch-activated reflexes in vascular epithelium and smooth muscle; gravitaxis and turgor control in plants; tissue development and morphogenesis; and the senses of touch, hearing, and balance. Intense genetic, molecular, and elecrophysiological studies in organisms ranging from nematodes to mammals have highlighted members of the recently discovered DEG/ENaC family of ion channels as strong candidates for the elusive metazoan mechanotransducer. Here, we discuss the evidence that links DEG/ENaC ion channels to mechanotransduction and review the function of Caenorhabiditis elegans members of this family called degenerins and their role in mediating mechanosensitive behaviors in the worm.     

Plants as Environmental Biosensors

Plants are continuously exposed to a wide variety of perturbations including variation of temperature and/or light, mechanical forces, gravity, air and soil pollution, drought, deficiency or surplus of nutrients, attacks by insects and pathogens, etc., and hence, it is essential for all plants to have survival sensory mechanisms against such perturbations. Consequently, plants generate various types of intracellular and intercellular electrical signals mostly in the form of action and variation potentials in response to these environmental changes. However, over a long period, only certain plants with rapid and highly noticeable responses for environmental stresses have received much attention from plant scientists. Of particular interest to our recent studies on ultra fast action potential measurements in green plants, we discuss in this review the evidence supporting the foundation for utilizing green plants as fast biosensors for molecular recognition of the direction of light, monitoring the environment, and detecting the insect attacks as well as the effects of pesticides, defoliants, uncouplers, and heavy metal pollutants.Key Words: plant signaling, plant electrophysiology, action potential, biosensor     

Arabidopsis TCH3 encodes a novel Ca2+ binding protein and shows environmentally induced and tissue-specific regulation.

The Arabidopsis touch (TCH) genes are up-regulated in response to various environmental stimuli, including touch, wind, and darkness. Previously, it was determined that TCH1 encodes a calmodulin; TCH2 and TCH3 encode calmodulin-related proteins. Here, we present the sequence and genomic organization of TCH3. TCH3 is composed of three repeats; remarkably, the first two repeats share 94% sequence identity, including introns that are 99% identical. The conceptual TCH3 product is 58 to 60% identical to known Arabidopsis calmodulins; however, unlike calmodulin, which has four Ca2+ binding sites, TCH3 has six potential Ca2+ binding domains. TCH3 is capable of binding Ca2+, as demonstrated by a Ca(2+)-specific shift in electrophoretic mobility. 5' Fragments of the TCH3 locus, when fused to the beta-glucuronidase (GUS) reporter gene, are sufficient to confer inducibility of expression following stimulation of plants with touch or darkness. These TCH3 sequences also direct expression to growing regions of roots, vascular tissue, root/shoot junctions, trichomes, branch points of the shoot, and regions of siliques and flowers. The pattern of expression of the TCH3/GUS reporter genes most likely reflects expression of the native TCH3 gene, because immunostaining of the TCH3 protein shows similar localization. The tissue-specific expression of TCH3 suggests that expression may be regulated not only by externally applied mechanical stimuli but also by mechanical stresses generated during development. Consequently, TCH3 may perform a Ca(2+)-modulated function involved in generating changes in cells and/or tissues that result in greater strength or flexibility.