Temperature changes in the root ecosystem affect plant functionality.

Climate change is increasing the frequency of extreme heat events that aggravate its negative impact on plant development and agricultural yield. Most experiments designed to study plant adaption to heat stress apply homogeneous high temperatures to both shoot and root. However, this treatment does not mimic the conditions in natural fields, where roots grow in a dark environment with a descending temperature gradient. Excessively high temperatures severely decrease cell division in the root meristem, compromising root growth, while increasing the division of quiescent center cells, likely in an attempt to maintain the stem cell niche under such harsh conditions. Here, we engineered the TGRooZ, a device that generates a temperature gradient for in vitro or greenhouse growth assays. The root systems of plants exposed to high shoot temperatures but cultivated in the TGRooZ grow efficiently and maintain their functionality to sustain proper shoot growth and development. Furthermore, gene expression and rhizosphere or root microbiome composition are significantly less affected in TGRooZ-grown roots than in high-temperature-grown roots, correlating with higher root functionality. Our data indicate that use of the TGRooZ in heat-stress studies can improve our knowledge of plant response to high temperatures, demonstrating its applicability from laboratory studies to the field.

Alternative Polyadenylation and Salicylic Acid Modulate Root Responses to Low Nitrogen Availability.

Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5'-UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

Role of cis-zeatin in root responses to phosphate starvation.

Phosphate (Pi) is an essential nutrient for all organisms. Roots are underground organs, but the majority of the root biology studies have been done on root systems growing in the presence of light. Root illumination alters the Pi starvation response (PSR) at different intensities. Thus, we have analyzed morphological, transcriptional and physiological responses to Pi starvation in dark-grown roots. We have identified new genes and pathways regulated by Pi starvation that were not described previously. We also show that Pi-starved plants increase the cis-zeatin (cZ) : trans-zeatin (tZ) ratio. Transcriptomic analyses show that tZ preferentially represses cell cycle and PSR genes, whereas cZ induces genes involved in cell and root hair elongation and differentiation. In fact, cZ-treated seedlings show longer root system as well as longer root hairs compared with tZ-treated seedlings, increasing the total absorbing surface. Mutants with low cZ concentrations do not allocate free Pi in roots during Pi starvation. We propose that Pi-starved plants increase the cZ : tZ ratio to maintain basal cytokinin responses and allocate Pi in the root system to sustain its growth. Therefore, cZ acts as a PSR hormone that stimulates root and root hair elongation to enlarge the root absorbing surface and to increase Pi concentrations in roots.

The polyadenylation factor FIP1 is important for plant development and root responses to abiotic stresses.

Root development and its response to environmental changes is crucial for whole plant adaptation. These responses include changes in transcript levels. Here, we show that the alternative polyadenylation (APA) of mRNA is important for root development and responses. Mutations in FIP1, a component of polyadenylation machinery, affects plant development, cell division and elongation, and response to different abiotic stresses. Salt treatment increases the amount of poly(A) site usage within the coding region and 5' untranslated regions (5'-UTRs), and the lack of FIP1 activity reduces the poly(A) site usage within these non-canonical sites. Gene ontology analyses of transcripts displaying APA in response to salt show an enrichment in ABA signaling, and in the response to stresses such as salt or cadmium (Cd), among others. Root growth assays show that fip1-2 is more tolerant to salt but is hypersensitive to ABA or Cd. Our data indicate that FIP1-mediated alternative polyadenylation is important for plant development and stress responses.

A light-sensitive mutation in Arabidopsis LEW3 reveals the important role of N-glycosylation in root growth and development.

Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.

The transcription factor OBP4 controls root growth and promotes callus formation.

Plant growth and development require a continuous balance between cell division and differentiation. In root meristems, differentiated cells acquire specialized functions, losing their mitotic potential. Some plant cells, such as pericycle cells, have a remarkable plasticity to regenerate new organs. The molecular mechanisms underlying cell reprogramming are not completely known. In this work, a functional screening of transcription factors identified Arabidopsis OBP4 (OBF Binding Protein 4) as a novel regulator of root growth and cell elongation and differentiation. Overexpression of OBP4 regulates the levels of a large number of transcripts in roots, many involved in hormonal signaling and callus formation. OBP4 controls cell elongation and differentiation in root cells. OBP4 does not induce cell division in the root meristem, but promotes pericycle cell proliferation, forming callus-like structures at the root tip, as shown by the expression of stem cell markers. Callus formation is enhanced by ectopic expression of OBP4 in the wild-type or alf4-1, but is significantly reduced in roots that have lower levels of OBP4. Our data provide molecular insights into how differentiated root cells acquire the potential to generate callus, a pluripotent mass of cells that can regenerate fully functional plant organs.

Flavonols Mediate Root Phototropism and Growth through Regulation of Proliferation-to-Differentiation Transition.

Roots normally grow in darkness, but they may be exposed to light. After perceiving light, roots bend to escape from light (root light avoidance) and reduce their growth. How root light avoidance responses are regulated is not well understood. Here, we show that illumination induces the accumulation of flavonols in Arabidopsis thaliana roots. During root illumination, flavonols rapidly accumulate at the side closer to light in the transition zone. This accumulation promotes asymmetrical cell elongation and causes differential growth between the two sides, leading to root bending. Furthermore, roots illuminated for a long period of time accumulate high levels of flavonols. This high flavonol content decreases both auxin signaling and PLETHORA gradient as well as superoxide radical content, resulting in reduction of cell proliferation. In addition, cytokinin and hydrogen peroxide, which promote root differentiation, induce flavonol accumulation in the root transition zone. As an outcome of prolonged light exposure and flavonol accumulation, root growth is reduced and a different root developmental zonation is established. Finally, we observed that these differentiation-related pathways are required for root light avoidance. We propose that flavonols function as positional signals, integrating hormonal and reactive oxygen species pathways to regulate root growth direction and rate in response to light.

D-Root: a system for cultivating plants with the roots in darkness or under different light conditions.

In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark-Root (D-Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D-Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D-Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.