Not so hidden anymore: Advances and challenges in understanding root growth under water deficits.

Limited water availability is a major environmental factor constraining plant development and crop yields. One of the prominent adaptations of plants to water deficits is the maintenance of root growth that enables sustained access to soil water. Despite early recognition of the adaptive significance of root growth maintenance under water deficits, progress in understanding has been hampered by the inherent complexity of root systems and their interactions with the soil environment. We highlight selected milestones in the understanding of root growth responses to water deficits, with emphasis on founding studies that have shaped current knowledge and set the stage for further investigation. We revisit the concept of integrated biophysical and metabolic regulation of plant growth and use this framework to review central growth-regulatory processes occurring within root growth zones under water stress at subcellular to organ scales. Key topics include the primary processes of modifications of cell wall-yielding properties and osmotic adjustment, as well as regulatory roles of abscisic acid and its interactions with other hormones. We include consideration of long-recognized responses for which detailed mechanistic understanding has been elusive until recently, for example hydrotropism, and identify gaps in knowledge, ongoing challenges, and opportunities for future research.

Ethylene inhibits rice root elongation in compacted soil via ABA- and auxin-mediated mechanisms.

Soil compaction represents a major agronomic challenge, inhibiting root elongation and impacting crop yields. Roots use ethylene to sense soil compaction as the restricted air space causes this gaseous signal to accumulate around root tips. Ethylene inhibits root elongation and promotes radial expansion in compacted soil, but its mechanistic basis remains unclear. Here, we report that ethylene promotes abscisic acid (ABA) biosynthesis and cortical cell radial expansion. Rice mutants of ABA biosynthetic genes had attenuated cortical cell radial expansion in compacted soil, leading to better penetration. Soil compaction-induced ethylene also up-regulates the auxin biosynthesis gene OsYUC8. Mutants lacking OsYUC8 are better able to penetrate compacted soil. The auxin influx transporter OsAUX1 is also required to mobilize auxin from the root tip to the elongation zone during a root compaction response. Moreover, osaux1 mutants penetrate compacted soil better than the wild-type roots and do not exhibit cortical cell radial expansion. We conclude that ethylene uses auxin and ABA as downstream signals to modify rice root cell elongation and radial expansion, causing root tips to swell and reducing their ability to penetrate compacted soil.

A multiple ion-uptake phenotyping platform reveals shared mechanisms affecting nutrient uptake by roots.

Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high-throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion-uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that uptake is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.

Bioinformatics for plant and agricultural discoveries in the age of multiomics: A review and case study of maize nodal root growth under water deficit.

Advances in next-generation sequencing and other high-throughput technologies have facilitated multiomics research, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, and phenomics. The resultant emerging multiomics data have brought new challenges as well as opportunities, as seen in the plant and agriculture science domains. We reviewed several bioinformatic and computational methods, models, and platforms, and we have highlighted some of our in-house developed efforts aimed at multiomics data analysis, integration, and management issues faced by the research community. A case study using multiomics datasets generated from our studies of maize nodal root growth under water deficit stress demonstrates the power of these datasets and some other publicly available tools. This analysis also sheds light on the landscape of such applied bioinformatic tools currently available for plant and crop science studies and introduces emerging trends and how they may affect the future.

Maize lateral root developmental plasticity induced by mild water stress. II: Genotype-specific spatio-temporal effects on determinate development.

Maize lateral roots exhibit determinate growth, whereby the meristem is genetically programmed to stop producing new cells. To explore whether lateral root determinacy is modified under water deficits, we studied two maize genotypes (B73 and FR697) with divergent responses of lateral root growth to mild water stress using an experimental system that provided near-stable water potential environments throughout lateral root development. First-order laterals of the primary root system of FR697 exhibited delayed determinacy when grown at a water potential of -0.28 MPa, resulting in longer and wider roots than in well-watered (WW) controls. In B73, in contrast, neither the length nor width of lateral roots was affected by water deficit. In water-stressed FR697, root elongation continued at or above the maximum rate in WW roots for 3 days longer, and was still 45% of maximum when WW roots approached their determinate length. Maintenance of root elongation was associated with sustained rates of cell production. In addition, kinematic analyses showed that reductions in tissue expansion rates with aging were delayed in the longitudinal, radial and tangential planes throughout the root growth zone. Thus, this study reveals large genotypic differences in the interaction of water stress with developmental determinacy of maize lateral roots.

Water-deficit responsive microRNAs in the primary root growth zone of maize.

BACKGROUND: MicroRNA-mediated gene regulatory networks play a significant role in plant growth and development and environmental stress responses. RESULTS: We identified 79 microRNAs (miRNAs) and multiple miRNA variants (isomiRs) belonging to 26 miRNA families in the primary root growth zone of maize seedlings grown at one of three water potentials: well-watered (- 0.02 MPa), mild water deficit stress (- 0.3 MPa), and severe water deficit stress (- 1.6 MPa). The abundances of 3 miRNAs (mild stress) and 34 miRNAs representing 17 families (severe stress) were significantly different in water-deficit stressed relative to well-watered controls (FDR < 0.05 and validated by stem loop RT-qPCR). Degradome sequencing revealed 213 miRNA-regulated transcripts and trancriptome profiling revealed that the abundance of 77 (miRNA-regulated) were regulated by water-defecit stress. miR399e,i,j-3p was strongly regulated by water-defcit stress implicating the possibility of nutrient deficiency during stress. CONCLUSIONS: We have identified a number of maize miRNAs that respond to specific water deficits applied to the primary root growth zone. We have also identified transcripts that are targets for miRNA regulation in the root growth zone under water-deficit stress. The miR399e,i,j-3p that is known to regulate phosphate uptake in response to nutrient deficiencies responds to water-deficit stress, however, at the seedling stage the seed provides adequate nutrients for root growth thus miR399e,i,j-3p may play a separate role in water-deficit responses. A water-deficit regulated maize transcript, similar to known miR399 target mimics, was identified and we hypothesized that it is another regulatory player, moderating the role of miR399e,i,j-3p, in primary root growth zone water deficit responses.

Maize lateral root developmental plasticity induced by mild water stress. I: Genotypic variation across a high-resolution series of water potentials.

Lateral root developmental plasticity induced by mild water stress was examined across a high-resolution series of growth media water potentials (Ψw ) in two genotypes of maize. The suitability of several media for imposing near-stable Ψw treatments on transpiring plants over prolonged growth periods was assessed. Genotypic differences specific to responses of lateral root growth from the primary root system occurred between cultivars FR697 and B73 over a narrow series of water stress treatments ranging in Ψw from -0.25 to -0.40 MPa. In FR697, both the average length and number of first-order lateral roots were substantially enhanced at a Ψw of -0.25 MPa compared with well-watered controls. These effects were separated spatially, occurring primarily in the upper and lower regions of the axial root, respectively. Furthermore, first-order lateral roots progressively increased in diameter with increasing water stress, resulting in a maximum 2.3-fold increase in root volume at a Ψw of -0.40 MPa. In B73, in contrast, the length, diameter, nor number of lateral roots was increased in any of the water stress treatments. The genotype-specific responses observed over this narrow range of Ψw demonstrate the necessity of high-resolution studies at mild stress levels for characterization of lateral root developmental plasticity.

Water-deficit-induced changes in transcription factor expression in maize seedlings.

Plants tolerate water deficits by regulating gene networks controlling cellular and physiological traits to modify growth and development. Transcription factor (TF)-directed regulation of transcription within these gene networks is key to eliciting appropriate responses. In this study, reverse transcription quantitative PCR (RT-qPCR) was used to examine the abundance of 618 transcripts from 536 TF genes in individual root and shoot tissues of maize seedlings grown in vermiculite under well-watered (water potential of -0.02 MPa) and water-deficit conditions (water potentials of -0.3 and -1.6 MPa). A linear mixed model identified 433 TF transcripts representing 392 genes that differed significantly in abundance in at least one treatment, including TFs that intersect growth and development and environmental stress responses. TFs were extensively differentially regulated across stressed maize seedling tissues. Hierarchical clustering revealed TFs with stress-induced increased abundance in primary root tips that likely regulate root growth responses to water deficits, possibly as part of abscisic acid and/or auxin-dependent signaling pathways. Ten of these TFs were selected for validation in nodal root tips of drought-stressed field-grown plants (late V1 to early V2 stage). Changes in abundance of these TF transcripts under a field drought were similar to those observed in the seedling system.

Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

Plate-based diversity subset screening generation 2: an improved paradigm for high-throughput screening of large compound files.

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.

Physiological responses related to increased grain yield under drought in the first biotechnology-derived drought-tolerant maize.

Maize (Zea mays ssp. mays L.) is highly susceptible to drought stress. This work focused on whole-plant physiological mechanisms by which a biotechnology-derived maize event expressing bacterial cold shock protein B (CspB), MON 87460, increased grain yield under drought. Plants of MON 87460 and a conventional control (hereafter 'control') were tested in the field under well-watered (WW) and water-limited (WL) treatments imposed during mid-vegetative to mid-reproductive stages during 2009-2011. Across years, average grain yield increased by 6% in MON 87460 compared with control under WL conditions. This was associated with higher soil water content at 0.5 m depth during the treatment phase, increased ear growth, decreased leaf area, leaf dry weight and sap flow rate during silking, increased kernel number and harvest index in MON 87460 than the control. No consistent differences were observed under WW conditions. This indicates that MON 87460 acclimated better under WL conditions than the control by lowering leaf growth which decreased water use during silking, thereby eliciting lower stress under WL conditions. These physiological responses in MON 87460 under WL conditions resulted in increased ear growth during silking, which subsequently increased the kernel number, harvest index and grain yield compared to the control.

Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean.

Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include functional genes and regulatory transcription factors. The Arabidopsis thaliana DREB1D transcription factor driven by the constitutive and ABA-inducible promoters was introduced into soybean through Agrobacterium tumefaciens-mediated gene transfer. Several transgenic lines were generated and molecular analysis was performed to confirm transgene integration. Transgenic plants with an ABA-inducible promoter showed a 1.5- to two-fold increase of transgene expression under severe stress conditions. Under well-watered conditions, transgenic plants with constitutive and ABA-inducible promoters showed reduced total leaf area and shoot biomass compared to non-transgenic plants. No significant differences in root length or root biomass were observed between transgenic and non-transgenic plants under non-stress conditions. When subjected to gradual water deficit, transgenic plants maintained higher relative water content because the transgenic lines used water more slowly as a result of reduced total leaf area. This caused them to wilt slower than non-transgenic plants. Transgenic plants showed differential drought tolerance responses with a significantly higher survival rate compared to non-transgenic plants when subjected to comparable severe water-deficit conditions. Moreover, the transgenic plants also showed improved drought tolerance by maintaining 17-24 % greater leaf cell membrane stability compared to non-transgenic plants. The results demonstrate the feasibility of engineering soybean for enhanced drought tolerance by expressing stress-responsive genes.

Apoplastic hydrogen peroxide in the growth zone of the maize primary root under water stress. I. Increased levels are specific to the apical region of growth maintenance.

Previous work on the adaptation of maize (Zea mays L.) primary root growth to water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. Cell wall proteomic analysis suggested that levels of apoplastic reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), may be modified in a region-specific manner within the growth zone of water-stressed roots. Apoplastic ROS may have wall loosening or tightening effects and may also have other growth regulatory functions. To gain an understanding of how apoplastic ROS levels change under water stress, cerium chloride staining was used in conjunction with transmission electron microscopy to examine the spatial distribution of apoplastic H2O2. The results revealed that apoplastic H2O2 levels increased specifically in the apical region of the growth zone under water stress, correlating spatially with the maintenance of cell elongation. The basal regions of the growth zone of water-stressed roots and the entire growth zone of well-watered roots exhibited relatively low levels of apoplastic H2O2. The increase in apoplastic H2O2 in the apical region under water stress probably resulted, at least in part, from a pronounced increase in oxalate oxidase activity in this region. By contrast, well-watered roots showed negligible oxalate oxidase activity throughout the growth zone. The results show that changes in apoplastic ROS levels in the root growth zone under water-deficit conditions are regulated in a spatially-specific manner, suggesting that this response may play an important role in maize root adaptation to water stress.

Plate-based diversity subset screening: an efficient paradigm for high throughput screening of a large screening file.

The screening files of many large companies, including Pfizer, have grown considerably due to internal chemistry efforts, company mergers and acquisitions, external contracted synthesis, or compound purchase schemes. In order to screen the targets of interest in a cost-effective fashion, we devised an easy-to-assemble, plate-based diversity subset (PBDS) that represents almost the entire computed chemical space of the screening file whilst comprising only a fraction of the plates in the collection. In order to create this file, we developed new design principles for the quality assessment of screening plates: the Rule of 40 (Ro40) and a plate selection process that insured excellent coverage of both library chemistry and legacy chemistry space. This paper describes the rationale, design, construction, and performance of the PBDS, that has evolved into the standard paradigm for singleton (one compound per well) high-throughput screening in Pfizer since its introduction in 2006.

Comparative Transcriptomics Reveal Metabolic Rather than Genetic Control of Divergent Antioxidant Metabolism in the Primary Root Elongation Zone of Water-Stressed Cotton and Maize.

Under water stress, the primary root elongation zones of cotton and maize exhibit both conserved and divergent metabolic responses, including variations in sulfur and antioxidant metabolism. To explore the relative importance of metabolic and genetic controls of these responses for each species, and the extent to which responses are mediated by similar gene expression networks within the framework of ortholog groups, comparative transcriptomics analyses were conducted under conditions of equivalent tissue water stress. Ortholog analysis revealed that 86% of the transcriptome response to water stress was phylogenetically unrelated between cotton and maize. Elevated transcript abundances for genes involved in abscisic acid (ABA) biosynthesis and signaling, as well as key enzymes that enable osmotic adjustment, were conserved between the species. In contrast, antioxidant responses, at least with regard to glutathione metabolism and anti-oxidative enzymes, did not exhibit such a transcript abundance adaptive signature. In particular, previously characterized differential responses of the glutathione and sulfur metabolic pathways between cotton and maize were not evident in the transcriptomic responses. The findings indicate that the antioxidant response in both species results from a metabolic acclimation to water stress, and thus represents an example of water stress-related metabolic plasticity.

De novo transcriptome assembly from the nodal root growth zone of hydrated and water-deficit stressed maize inbred line FR697.

Certain cultivars of maize show increased tolerance to water deficit conditions by maintenance of root growth. To better understand the molecular mechanisms related to this adaptation, nodal root growth zone samples were collected from the reference inbred line B73 and inbred line FR697, which exhibits a relatively greater ability to maintain root elongation under water deficits. Plants were grown under various water stress levels in both field and controlled environment settings. FR697-specific RNA-Seq datasets were generated and used for a de novo transcriptome assembly to characterize any genotype-specific genetic features. The assembly was aided by an Iso-Seq library of transcripts generated from various FR697 plant tissue samples. The Necklace pipeline was used to combine a Trinity de novo assembly along with a reference guided assembly and the Viridiplantae proteome to generate an annotated consensus "SuperTranscriptome" assembly of 47,915 transcripts with a N50 of 3152 bp in length. The results were compared by Blastn to maize reference genes, a Benchmarking Universal Single-Copy Orthologs (BUSCO) genome completeness report and compared with three maize reference genomes. The resultant 'SuperTranscriptome' was demonstrated to be of high-quality and will serve as an important reference for analysis of the maize nodal root transcriptomic response to environmental perturbations.

Antioxidant Metabolism Underlies Different Metabolic Strategies for Primary Root Growth Maintenance under Water Stress in Cotton and Maize.

The divergence of metabolic responses to water stress in the elongation zone of cotton and maize primary roots was investigated by establishing water-deficit conditions that generated steady root elongation at equivalent tissue water potentials. In water-stressed cotton roots, cell elongation was maintained in the apical 3 mm but was progressively inhibited with further displacement from the apex. These responses are similar to previous findings in maize, providing the foundation for comparisons of metabolic responses in regions of growth maintenance and inhibition between the species. Metabolomics analyses showed region-specific and species-specific changes in metabolite abundance in response to water stress, revealing both conserved responses including osmolyte accumulation, and key differences in antioxidative and sulfur metabolism. Quantitative assessment showed contrasting glutathione responses in the root elongation zone between the species, with glutathione levels declining in cotton as stress duration progressed, whereas in maize, glutathione levels remained elevated. Despite the lesser glutathione response in cotton, hydrogen peroxide levels were low in water-stressed cotton compared with maize roots and were associated with higher catalase, ascorbate peroxidase, and superoxide dismutase activities in cotton. The results indicate alternative metabolic strategies underlying the responses of primary root growth to water stress between cotton and maize.

Water Deficit-Induced Changes in Phenolic Acid Content in Maize Leaves Is Associated with Altered Expression of Cinnamate 4-Hydroxylase and p-Coumaric Acid 3-Hydroxylase.

The amino acid phenylalanine is a precursor to phenolic acids that constitute the lignin biosynthetic pathway. Although there is evidence of a role of some phenolic acids in plant responses to pathogens and salinity, characterization of the involvement of phenolic acids in plant responses to drought is limited. Drought reduces water content in plant tissue and can lead to decreased cell viability and increased cell death. We thus subjected maize seedlings to water deficit and evaluated relative water content and cell viability together with p-coumaric acid, caffeic acid and ferulic acid contents in the leaves. Furthermore, we measured the enzymatic activity of cinnamate 4-hydroxylase (EC 1.14.13.11) and p-coumarate 3-hydroxylase (EC 1.14.17.2) and associated these with the expression of genes encoding cinnamate 4-hydroxylase and p-coumarate-3 hydroxylase in response to water deficit. Water deficit reduced relative water content and cell viability in maize leaves. This corresponded with decreased p-coumaric acid but increased caffeic and ferulic acid content in the leaves. Changes in the phenolic acid content of the maize leaves were associated with increased enzymatic activities of cinnamate 4-hydroxylase and p-coumarate hydroxylase. The increased enzymatic activity of p-coumarate 3-hydroxylase was associated with increased expression of a gene encoding p-coumarate 3-hydroxylase. We thus conclude that metabolic pathways involving phenolic acids may contribute to the regulation of drought responses in maize, and we propose that further work to elucidate this regulation may contribute to the development of new maize varieties with improved drought tolerance. This can be achieved by marker-assisted selection to select maize lines with high levels of expression of genes encoding cinnamate 4-hydroxylase and/or p-coumarate 3-hydroxylase for use in breeding programs aimed and improving drought tolerance, or by overexpression of these genes via genetic engineering to confer drought tolerance.

Hydraulic flux-responsive hormone redistribution determines root branching.

Plant roots exhibit plasticity in their branching patterns to forage efficiently for heterogeneously distributed resources, such as soil water. The xerobranching response represses lateral root formation when roots lose contact with water. Here, we show that xerobranching is regulated by radial movement of the phloem-derived hormone abscisic acid, which disrupts intercellular communication between inner and outer cell layers through plasmodesmata. Closure of these intercellular pores disrupts the inward movement of the hormone signal auxin, blocking lateral root branching. Once root tips regain contact with moisture, the abscisic acid response rapidly attenuates. Our study reveals how roots adapt their branching pattern to heterogeneous soil water conditions by linking changes in hydraulic flux with dynamic hormone redistribution.

Complexity and coordination of root growth at low water potentials: recent advances from transcriptomic and proteomic analyses.

Progress in understanding root growth regulation and adaptation under water-stressed conditions is reviewed, with emphasis on recent advances from transcriptomic and proteomic analyses of maize and soybean primary roots. In both systems, kinematic characterization of the spatial patterns of cell expansion within the root elongation zone showed that at low water potentials, elongation rates are preferentially maintained towards the root apex but are progressively inhibited at more basal locations resulting in a shortened growth zone. This characterization provided an essential foundation for extensive research into the physiological mechanisms of growth regulation in the maize primary root at low water potentials. Recently, these studies were expanded to include transcriptomic and cell wall proteomic analyses of the maize primary root, and a proteomic analysis of total soluble proteins in the soybean primary root. This review focuses on findings related to protection from oxidative damage, the potential roles of increased apoplastic reactive oxygen species in regulation of wall extension properties and other processes, region-specific phenylpropanoid metabolism as related to accumulation of (iso)flavonoids and wall phenolics and amino acid metabolism. The results provide novel insights into the complexity and coordination of the processes involved in root growth at low water potentials.