Janet Braam

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.

Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis thaliana

Phytotoxicity is an important consideration to understand the potential environmental impacts of manufactured nanomaterials. Here, we report on the effects of four metal oxide nanoparticles, aluminum oxide (nAl(2)O(3)), silicon dioxide (nSiO(2)), magnetite (nFe(3)O(4)), and zinc oxide (nZnO), on the development of Arabidopsis thaliana (Mouse-ear cress). Three toxicity indicators (seed germination, root elongation, and number of leaves) were quantified following exposure to each nanoparticle at three concentrations: 400, 2,000, and 4,000 mg/L. Among these particles, nZnO was most phytotoxic, followed by nFe(3)O(4), nSiO(2), and nAl(2)O(3), which was not toxic. Consequently, nZnO was further studied to discern the importance of particle size and zinc dissolution as toxicity determinants. Soluble zinc concentrations in nanoparticle suspensions were 33-fold lower than the minimum inhibitory concentration of dissolved zinc salt (ZnCl(2)), indicating that zinc dissolution could not solely account for the observed toxicity. Inhibition of seed germination by ZnO depended on particle size, with nanoparticles exerting higher toxicity than larger (micron-sized) particles at equivalent concentrations. Overall, this study shows that direct exposure to nanoparticles significantly contributed to phytotoxicity and underscores the need for eco-responsible disposal of wastes and sludge containing metal oxide nanoparticles.

Xyloglucan endotransglycosylases: diversity of genes, enzymes and potential wall-modifying functions

Plant cells are enclosed by walls that define the shapes and sizes of cells and mediate cell-to-cell contact. The dynamics of plant growth, morphogenesis and differentiation require concomitant modifications of the walls. A class of enzymes known as xyloglucan endotransglycosylases have the potential to enzymatically modify wall components, but although their biochemical activity has been defined, the physiological roles of xyloglucan endotransglycosylases remain undefined. Xyloglucan endotransglycosylases are encoded by large gene families, and in an attempt to clarify their physiological role, the diverse regulation of the genes and properties of the proteins are being determined.

Cold-Shock Regulation of the Arabidopsis TCH Genes and the Effects of Modulating Intracellular Calcium Levels

The Arabidopsis TCH genes, which encode calmodulin-related proteins and a xyloglucan endotransglycosylase, are shown to be up-regulated in expression following cold shock. We investigated a possible role of fluctuations in intracellular calcium ion concentra- tions ([Ca2+]) in the cold-shock-induced TCH gene expression. Transgenic plants harboring the apoaequorin gene were generated to monitor [Ca2+] and to test the necessity of cold-induced [Ca2+] increases for TCH expression. Cold-shock-induced [Ca2+] increases can be blocked by La3+ and Gd3+, putative plasma membrane Ca2+ channel blockers, and 1,2-bis(o-aminophenoxy)ethane-N,N,N[prime],N[prime]- tetraacetic acid, an extracellular Ca2+ chelator. Cold-shock-induced expression of the TCH genes is inhibited by levels of La3+, Gd3+, and 1,2-bis(o-aminophenoxy)ethane-N,N,N[prime],N[prime]-tetraacetic acid, that have been shown to block [Ca2+] increases. These data support the hypotheses that (a) intracellular [Ca2+] increases following cold shock require extracellular Ca2+ and may derive from a Ca2+ influx mediated by plasmalemma Ca2+ channels, and (b) cold up-regulation of expression of at least a subset of the TCH genes requires an intracellular [Ca2+] increase. The inhibitors are also shown to have stimulus-independent effects on gene expression, providing strong evidence that these commonly used chemicals have more complex effects than generally reported.

Thigmomorphogenesis: a complex plant response to mechano-stimulation

In nature, plants are challenged with hurricane winds, monsoon rains, and herbivory attacks, in addition to many other harsh mechanical perturbations that can threaten plant survival. As a result, over many years of evolution, plants have developed very sensitive mechanisms through which they can perceive and respond to even subtle stimuli, like touch. Some plants respond behaviourally to the touch stimulus within seconds, while others show morphogenetic alterations over long periods of time, ranging from days to weeks. Various signalling molecules and phytohormones, including intracellular calcium, jasmonates, ethylene, abscisic acid, auxin, brassinosteroids, nitric oxide, and reactive oxygen species, have been implicated in touch responses. Many genes are induced following touch. These genes encode proteins involved in various cellular processes including calcium sensing, cell wall modifications, and defence. Twenty-three per cent of these up-regulated genes contain a recently identified promoter element involved in the rapid induction in transcript levels following mechanical perturbations. The employment of various genetic, biochemical, and molecular tools may enable elucidation of the mechanisms through which plants perceive mechano-stimuli and transduce the signals intracellularly to induce appropriate responses.

Innate immunity signaling: cytosolic Ca2+ elevation is linked to downstream nitric oxide generation through the action of calmodulin or a calmodulin-like protein.

Ca2+ rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca2+-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca2+ rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca2+/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca2+ signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca2+ elevation, excluding the possibility of CaM acting upstream from Ca2+. The CaM antagonist and Ca2+ chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca2+/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca2+ influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.

Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior

Diverse life forms have evolved internal clocks enabling them to monitor time and thereby anticipate the daily environmental changes caused by Earths rotation. The plant circadian clock regulates expression of about one-third of the Arabidopsis genome, yet the physiological relevance of this regulation is not fully understood. Here we show that the circadian clock, acting with hormone signals, provides selective advantage to plants through anticipation of and enhanced defense against herbivory. We found that cabbage loopers (Trichoplusia ni) display rhythmic feeding behavior that is sustained under constant conditions, and plants entrained in light/dark cycles coincident with the entrainment of the T. ni suffer only moderate tissue loss due to herbivory. In contrast, plants entrained out-of-phase relative to the insects are significantly more susceptible to attack. The in-phase entrainment advantage is lost in plants with arrhythmic clocks or deficient in jasmonate hormone; thus, both the circadian clock and jasmonates are required. Circadian jasmonate accumulation occurs in a phase pattern consistent with preparation for the onset of peak circadian insect feeding behavior, providing evidence for the underlying mechanism of clock-enhanced herbivory resistance. Furthermore, we find that salicylate, a hormone involved in biotrophic defense that often acts antagonistically to jasmonates, accumulates in opposite phase to jasmonates. Our results demonstrate that the plant circadian clock provides a strong physiological advantage by performing a critical role in Arabidopsis defense.

CML24, Regulated in Expression by Diverse Stimuli, Encodes a Potential Ca2+ Sensor That Functions in Responses to Abscisic Acid, Daylength, and Ion Stress

Changes in intracellular calcium (Ca2+) levels serve to signal responses to diverse stimuli. Ca2+ signals are likely perceived through proteins that bind Ca2+, undergo conformation changes following Ca2+ binding, and interact with target proteins. The 50-member calmodulin-like (CML) Arabidopsis (Arabidopsis thaliana) family encodes proteins containing the predicted Ca2+-binding EF-hand motif. The functions of virtually all these proteins are unknown. CML24, also known as TCH2, shares over 40% amino acid sequence identity with calmodulin, has four EF hands, and undergoes Ca2+-dependent changes in hydrophobic interaction chromatography and migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs, and transcript levels are increased from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA), and indole-3-acetic acid. However, CML24 protein accumulation changes were not detectable. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress; in regions undergoing growth; in vascular tissues and various floral organs; and in stomata, trichomes, and hydathodes. CML24-underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, are defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4, and MgCl2. MgCl2 tolerance is not due to reduced uptake or to elevated Ca2+ accumulation. Together, these data present evidence that CML24, a gene expressed in diverse organs and responsive to diverse stimuli, encodes a potential Ca2+ sensor that may function to enable responses to ABA, daylength, and presence of various salts.

Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation.

A gene family encoding xyloglucan endotransglycosylase (XET)-related proteins exists in Arabidopsis. TCH4, a member of this family, is strongly up-regulated by environmental stimuli and encodes an XET capable of modifying cell wall xyloglucans. To investigate XET localization we generated antibodies against the TCH4 carboxyl terminus. The antibodies recognized TCH4 and possibly other XET-related proteins. These data indicate that XETs accumulate in expanding cells, at the sites of intercellular airspace formation, and at the bases of leaves, cotyledons, and hypocotyls. XETs also accumulated in vascular tissue, where cell wall modifications lead to the formation of tracheary elements and sieve tubes. Thus, XETs may function in modifying cell walls to allow growth, airspace formation, the development of vasculature, and reinforcement of regions under mechanical strain. Following wind stimulation, overall XET levels appeared to decrease in the leaves of wind-stimulated plants. However, consistent with an increase in TCH4 mRNA levels following wind, there were regions that showed increased immunoreaction, including sites around cells of the pith parenchyma, between the vascular elements, and within the epidermis. These results indicate that TCH4 may contribute to the adaptive changes in morphogenesis that occur in Arabidopsis following exposure to mechanical stimuli.

Phytostimulation of Poplars and Arabidopsis Exposed to Silver Nanoparticles and Ag+ at Sublethal Concentrations

The increasing likelihood of silver nanoparticle (AgNP) releases to the environment highlights the importance of understanding AgNP interactions with plants, which are cornerstones of most ecosystems. In this study, poplars (Populus deltoides × nigra) and Arabidopsis thaliana were exposed hydroponically to nanoparticles of different sizes (PEG-coated 5 and 10 nm AgNPs, and carbon-coated 25 nm AgNPs) or silver ions (Ag(+), added as AgNO₃) at a wide range of concentrations (0.01 to 100 mg/L). Whereas all forms of silver were phytotoxic above a specific concentration, a stimulatory effect was observed on root elongation, fresh weight, and evapotranspiration of both plants at a narrow range of sublethal concentrations (e.g., 1 mg/L of 25 nm AgNPs for poplar). Plants were most susceptible to the toxic effects of Ag(+) (1 mg/L for poplar, 0.05 mg/L for Arabidopsis), but AgNPs also showed some toxicity at higher concentrations (e.g., 100 mg/L of 25 nm AgNPs for poplar, 1 mg/L of 5 nm AgNPs for Arabidopsis) and this susceptibility increased with decreasing AgNP size. Both poplars and Arabidopsis accumulated silver, but silver distribution in shoot organs varied between plant species. Arabidopsis accumulated silver primarily in leaves (at 10-fold higher concentrations than in the stem or flower tissues), whereas poplars accumulated silver at similar concentrations in leaves and stems. Within the particle subinhibitory concentration range, silver accumulation in poplar tissues increased with exposure concentration and with smaller AgNP size. However, compared to larger AgNPs, the faster silver uptake associated with smaller AgNPs was offset by their toxic effect on evapotranspiration, which was exerted at lower concentrations (e.g., 1 mg/L of 5 nm AgNPs for poplar). Overall, the observed phytostimulatory effects preclude generalizations about the phytotoxicity of AgNPs and encourage further mechanistic research.