Flexible Pectin Nanopatterning Drives Cell Wall Organization in Plants.

Plant cell walls are abundant sources of materials and energy. Nevertheless, cell wall nanostructure, specifically how pectins interact with cellulose and hemicelluloses to construct a robust and flexible biomaterial, is poorly understood. X-ray scattering measurements are minimally invasive and can reveal ultrastructural, compositional, and physical properties of materials. Resonant X-ray scattering takes advantage of compositional differences by tuning the energy of the incident X-ray to absorption edges of specific elements in a material. Using Tender Resonant X-ray Scattering (TReXS) at the calcium K-edge to study hypocotyls of the model plant, Arabidopsis thaliana, we detected distinctive Ca features that we hypothesize correspond to previously unreported Ca-Homogalacturonan (Ca-HG) nanostructures. When Ca-HG structures were perturbed by chemical and enzymatic treatments, cellulose microfibrils were also rearranged. Moreover, Ca-HG nanostructure was altered in mutants with abnormal cellulose, pectin, or hemicellulose content. Our results indicate direct structural interlinks between components of the plant cell wall at the nanoscale and reveal mechanisms that underpin both the structural integrity of these components and the molecular architecture of the plant cell wall.

Measuring calcium content in plants using NEXAFS spectroscopy.

Calcium is important for the growth and development of plants. It serves crucial functions in cell wall and cell membrane structure and serves as a secondary messenger in signaling pathways relevant to nutrient and immunity responses. Thus, measuring calcium levels in plants is important for studies of plant biology and for technology development in food, agriculture, energy, and forest industries. Often, calcium in plants has been measured through techniques such as atomic absorption spectrophotometry (AAS), inductively coupled plasma-mass spectrometry (ICP-MS), and electrophysiology. These techniques, however, require large sample sizes, chemical extraction of samples or have limited spatial resolution. Here, we used near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the calcium L- and K-edges to measure the calcium to carbon mass ratio with spatial resolution in plant samples without requiring chemical extraction or large sample sizes. We demonstrate that the integrated absorbance at the calcium L-edge and the edge jump in the fluorescence yield at the calcium K-edge can be used to quantify the calcium content as the calcium mass fraction, and validate this approach with onion epidermal peels and ICP-MS. We also used NEXAFS to estimate the calcium mass ratio in hypocotyls of a model plant, Arabidopsis thaliana, which has a cell wall composition that is similar to that of onion epidermal peels. These results show that NEXAFS spectroscopy performed at the calcium edge provides an approach to quantify calcium levels within plants, which is crucial for understanding plant physiology and advancing plant-based materials.

Evidence for root adaptation to a spatially discontinuous water availability in the absence of external water potential gradients.

We hereby show that root systems adapt to a spatially discontinuous pattern of water availability even when the gradients of water potential across them are vanishingly small. A paper microfluidic approach allowed us to expose the entire root system of Brassica rapa plants to a square array of water sources, separated by dry areas. Gradients in the concentration of water vapor across the root system were as small as 10-4⋅mM⋅m-1 (∼4 orders of magnitude smaller than in conventional hydrotropism assays). Despite such minuscule gradients (which greatly limit the possible influence of the well-understood gradient-driven hydrotropic response), our results show that 1) individual roots as well as the root system as a whole adapt to the pattern of water availability to maximize access to water, and that 2) this adaptation increases as water sources become more rare. These results suggest that either plant roots are more sensitive to water gradients than humanmade water sensors by 3-5 orders of magnitude, or they might have developed, like other organisms, mechanisms for water foraging that allow them to find water in the absence of an external gradient in water potential.

Hydrogel-based transparent soils for root phenotyping in vivo.

Root phenotypes are increasingly explored as predictors of crop performance but are still challenging to characterize. Media that mimic field conditions (e.g., soil, sand) are opaque to most forms of radiation, while transparent media do not provide field-relevant growing conditions and phenotypes. We describe here a "transparent soil" formed by the spherification of hydrogels of biopolymers. It is specifically designed to support root growth in the presence of air, water, and nutrients, and allows the time-resolved phenotyping of roots in vivo by both photography and microscopy. The roots developed by soybean plants in this medium are significantly more similar to those developed in real soil than those developed in hydroponic conditions and do not show signs of hypoxia. Lastly, we show that the granular nature and tunable properties of these hydrogel beads can be leveraged to investigate the response of roots to gradients in water availability and soil stiffness.

Stress response to CO2 deprivation by Arabidopsis thaliana in plant cultures.

After being the standard plant propagation protocol for decades, cultures of Arabidopsis thaliana sealed with Parafilm remain common today out of practicality, habit, or necessity (as in co-cultures with microorganisms). Regardless of concerns over the aeration of these cultures, no investigation has explored the CO2 transport inside these cultures and its effect on the plants. Thereby, it was impossible to assess whether Parafilm-seals used today or in thousands of older papers in the literature constitute a treatment, and whether this treatment could potentially affect the study of other treatments.For the first time we report the CO2 concentrations in Parafilm-sealed cultures of A. thaliana with a 1 minute temporal resolution, and the transcriptome comparison with aerated cultures. The data show significant CO2 deprivation to the plants, a drastic suppression of photosynthesis, respiration, starch accumulation, chlorophyll biosynthesis, and an increased accumulation of reactive oxygen species. Most importantly, CO2 deprivation occurs as soon as the cotyledons emerge. Gene expression analysis indicates a significant alteration of 35% of the pathways when compared to aerated cultures, especially in stress response and secondary metabolism processes. On the other hand, the observed increase in the production of glucosinolates and flavonoids suggests intriguing possibilities for CO2 deprivation as an organic biofortification treatment in high-value crops.

From Petri Dishes to Model Ecosystems.

Model ecosystems could provide significant insight into the evolution and behavior of real ecosystems. We discuss the advantages and limitations of common approaches like mesocosms. In this context, we highlight recent breakthroughs that allow for the creation of networks of organisms with independently controlled environments and rates of chemical exchange.

HOMEs for plants and microbes - a phenotyping approach with quantitative control of signaling between organisms and their individual environments.

We describe a simple, scalable, modular, and frugal approach to create model ecosystems as millifluidic networks of interconnected habitats (hosting microbes or plants), which offers (i) quantitative and dynamic control over the exchange of chemicals between habitats, and (ii) independent control over their environment. Oscillatory laminar flows produce regions of vortex mixing around obstacles. When these overlap, rapid mass transport by dispersion occurs, which is quantitatively describable as diffusion, but is directional and tunable in rate over 3 orders of magnitude. This acceleration in the rate of diffusion is equivalent to reducing the distance between the habitats, and therefore, the organisms, down to the length scales characteristic of signaling in soil (<2 mm).

Simplicity as a Route to Impact in Materials Research.

Materials scientists and engineers desire to have an impact. In this Progress Report we postulate a close correlation between impact - whether academic, technological, or scientific - and simple solutions, here defined as solutions that are inexpensive, reliable, predictable, highly performing, "stackable" (i.e., they can be combined and compounded with little increase in complexity), and "hackable" (i.e., they can be easily modified and optimized). In light of examples and our own experience, we propose how impact can be pursued systematically in materials research through a simplicity-driven approach to discovery-driven or problem-driven research.

Plant Growth Environments with Programmable Relative Humidity and Homogeneous Nutrient Availability.

We describe the design, characterization, and use of "programmable", sterile growth environments for individual (or small sets of) plants. The specific relative humidities and nutrient availability experienced by the plant is established (RH between 15% and 95%; nutrient concentration as desired) during the setup of the growth environment, which takes about 5 minutes and <1$ in disposable cost. These systems maintain these environmental parameters constant for at least 14 days with minimal intervention (one minute every two days). The design is composed entirely of off-the-shelf components (e.g., LEGO® bricks) and is characterized by (i) a separation of root and shoot environment (which is physiologically relevant and facilitates imposing specific conditions on the root system, e.g., darkness), (ii) the development of the root system on a flat surface, where the root enjoys constant contact with nutrient solution and air, (iii) a compatibility with root phenotyping. We demonstrate phenotyping by characterizing root systems of Brassica rapa plants growing in different relative humidities (55%, 75%, and 95%). While most phenotypes were found to be sensitive to these environmental changes, a phenotype tightly associated with root system topology-the size distribution of the areas encircled by roots-appeared to be remarkably and counterintuitively insensitive to humidity changes. These setups combine many of the advantages of hydroponics conditions (e.g., root phenotyping, complete control over nutrient composition, scalability) and soil conditions (e.g., aeration of roots, shading of roots), while being comparable in cost and setup time to Magenta® boxes.

Phenotypic and molecular consequences of overexpression of metal-homeostasis genes.

Metal hyperaccumulating plants are able to store very large amounts of metals in their shoots. There are a number of reasons why it is important to be able to introduce metal hyperaccumulation traits into non-accumulating species (e.g., phytoremediation or biofortification in minerals) and to engineer a desired level of accumulation and distribution of metals. Metal homeostasis genes have therefore been used for these purposes. Engineered accumulation levels, however, have often been far from expected, and transgenic plants frequently display phenotypic features not related to the physiological function of the introduced gene. In this review, we focus on an aspect often neglected in research on plants expressing metal homeostasis genes: the specific regulation of endogenous metal homeostasis genes of the host plant in response to the transgene-induced imbalance of the metal status. These modifications constitute one of the major mechanisms involved in the generation of the plant's phenotype, including unexpected characteristics. Interestingly, activation of so-called "metal cross-homeostasis" has emerged as a factor of primary importance.

HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier.

Ectopic expression in tobacco (Nicotiana tabacum v. Xanthi) of the export protein AtHMA4 (responsible in Arabidopsis for the control of Zn/Cd root to shoot translocation) resulted in decreased Cd uptake/accumulation in roots and shoots. This study contributes to understanding the mechanisms underlying this Cd-dependent phenotype to help predict the consequences of transgene expression for potential phytoremediation/biofortification-based strategies. Microarray analysis was performed to identify metal homeostasis genes that were differentially expressed in roots of Cd-exposed AtHMA4-expressing tobacco relative to the wild type. It was established that down-regulation of genes known to mediate Cd uptake was not responsible for reduced Cd uptake/accumulation in AtHMA4 transformants. The transcript levels of NtIRT1 and NtZIP1 were higher in transgenic plants, indicating an induction of the Fe and Zn deficiency status due to AtHMA4 expression. Interestingly, upon exposure to Cd, genes involved in cell wall lignification (NtHCT, NtOMET, and NtPrx11a) were up-regulated in transformants. Microscopic analysis of roots demonstrated that expression of AtHMA4 caused an induction of cell wall lignification in the external cell layers that was accompanied by enhanced H2O2 accumulation. Further study showed that the concentration of other elements (B, Co, Cu, Ni, Mo, and Zn) was reduced in AtHMA4 transformants in the presence of Cd. In conclusion, due to ectopic expression of 35S::AtHMA4, the physical apoplastic barrier within the external cell layer developed, which is likely to be responsible for the reduction of Cd uptake/accumulation.

Development of Zn-related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Zn levels.

AtHMA4 was previously shown to contribute to the control of Zn root-to-shoot translocation and tolerance to high Zn. However, heterologous expression of 35S::AtHMA4 in tobacco (Nicotiana tabacum cv. Xanthi) results in enhanced Zn sensitivity. This study provides a better understanding of the development of this Zn-sensitive phenotype and demonstrates that substantial modifications of Zn homeostasis occur due to AtHMA4 expression. We show that ectopically expressing AtHMA4 in tobacco results in overloading the root and leaf apoplast with Zn. The tissue and cellular distribution of Zn, monitored using Zinpyr-1, was altered in the AtHMA4-expressing plants compared with wild type. Increased loading of the leaf apoplast with Zn in AtHMA4 transformants induced necrosis; this appeared at lower levels of Zn supply in the transgenics compared with wild type. This study suggests that Zn concentration may be sensed in the apoplast of leaves, and if concentrations are above a certain threshold then particular groups of cells accumulate Zn and necrosis is initiated. Therefore, this could be considered as a mechanism for protecting the other parts of the photosynthetically active leaf from Zn toxicity.

Expression of the P(1B) -type ATPase AtHMA4 in tobacco modifies Zn and Cd root to shoot partitioning and metal tolerance.

Genetic modification of Zn/Cd accumulation in roots and shoots for biofortification or phytoremediation is a focus of this manuscript. We expressed AtHMA4 (a P(1B) ATPase involved in Zn and Cd transport), AtHMA4-trunc (lacking the C-terminal region) and AtHMA4-C terminus (the C-terminal region alone) in tobacco under the CaMV 35S constitutive promoter and examined accumulation and tolerance to both metals. Expression of AtHMA4 enhanced Zn translocation to the shoots only at 10 µM Zn but not at 0.5, 100 and 200 µM Zn. AtHMA4-trunc did not show this effect and instead reduced Zn translocation to the shoot. AtHMA4-expressing plants showed a decrease in cadmium uptake when exposed to 0.25 and 5 µM Cd; this was also observed with AtHMA4-trunc-expressing lines, although to a lesser extent. Expression of AtHMA4-C-terminus containing potential metal binding sites increased cadmium and zinc concentrations in roots and shoots up to fourfold. We have demonstrated that both AtHMA4 and AtHMA4 C-terminus could be candidate genes/sequences for engineering modifications of zinc and cadmium root/shoot partitioning. However, the phenotype of transformants depended on the external metal concentration, thus it might be difficult to engineer a plant displaying the desired metal-related phenotype when grown under varying conditions of metal supply.

Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation.

Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications.