Root System Depth in Arabidopsis Is Shaped by EXOCYST70A3 via the Dynamic Modulation of Auxin Transport.

Root system architecture (RSA), the distribution of roots in soil, plays a major role in plant survival. RSA is shaped by multiple developmental processes that are largely governed by the phytohormone auxin, suggesting that auxin regulates responses of roots that are important for local adaptation. However, auxin has a central role in numerous processes, and it is unclear which molecular mechanisms contribute to the variation in RSA for environmental adaptation. Using natural variation in Arabidopsis, we identify EXOCYST70A3 as a modulator of the auxin system that causes variation in RSA by acting on PIN4 protein distribution. Allelic variation and genetic perturbation of EXOCYST70A3 lead to alteration of root gravitropic responses, resulting in a different RSA depth profile and drought resistance. Overall our findings suggest that the local modulation of the pleiotropic auxin pathway can gives rise to distinct RSAs that can be adaptive in specific environments.

ELONGATED HYPOCOTYL 5 regulates BRUTUS and affects iron acquisition and homeostasis in Arabidopsis thaliana.

Iron (Fe) is an essential micronutrient for both plants and animals. Fe-limitation significantly reduces crop yield and adversely impacts on human nutrition. Owing to limited bioavailability of Fe in soil, plants have adapted different strategies that not only regulate Fe-uptake and homeostasis but also bring modifications in root system architecture to enhance survival. Understanding the molecular mechanism underlying the root growth responses will have critical implications for plant breeding. Fe-uptake is regulated by a cascade of basic helix-loop-helix (bHLH) transcription factors (TFs) in plants. In this study, we report that HY5 (Elongated Hypocotyl 5), a member of the basic leucine zipper (bZIP) family of TFs, plays an important role in the Fe-deficiency signaling pathway in Arabidopsis thaliana. The hy5 mutant failed to mount optimum Fe-deficiency responses, and displayed root growth defects under Fe-limitation. Our analysis revealed that the induction of the genes involved in Fe-uptake pathway (FIT-FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR, FRO2-FERRIC REDUCTION OXIDASE 2 and IRT1-IRON-REGULATED TRANSPORTER1) is reduced in the hy5 mutant as compared with the wild-type plants under Fe-deficiency. Moreover, we also found that the expression of coumarin biosynthesis genes is affected in the hy5 mutant under Fe-deficiency. Our results also showed that HY5 negatively regulates BRUTUS (BTS) and POPEYE (PYE). Chromatin immunoprecipitation followed by quantitative polymerase chain reaction revealed direct binding of HY5 to the promoters of BTS, FRO2 and PYE. Altogether, our results showed that HY5 plays an important role in the regulation of Fe-deficiency responses in Arabidopsis.

HY5: a key regulator for light-mediated nutrient uptake and utilization by plants.

ELONGATED HYPOCOTYL 5 (HY5), a bZIP-type transcription factor, is a master regulator of light-mediated responses. ELONGATED HYPOCOTYL 5 binds to the promoter of c. 3000 genes, thereby regulating various physiological and biological processes, including photomorphogenesis, flavonoid biosynthesis, root development, response to abiotic stress and nutrient homeostasis. In recent decades, it has become clear that light signaling plays a crucial role in promoting nutrient uptake and assimilation. Recent studies have revealed the molecular mechanisms underlying such encouraging effects and the crucial function of the transcription factor HY5, whose activity is regulated by many photoreceptors. The discovery that HY5 directly activates the expression of genes involved in nutrient uptake and utilization, including several nitrogen, iron, sulphur, phosphorus and copper uptake and assimilation-related genes, enhances our understanding of how light signaling regulates uptake and utilisation of multiple nutrients in plants. Here, we review recent advances in the role of HY5 in light-dependent nutrient uptake and utilization.

Publisher Correction: An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

In this Letter, an incorrect version of the Supplementary Information file was inadvertently used, which contained several errors. The details of references 59-65 were missing from the end of the Supplementary Discussion section on page 4. In addition, the section 'Text 3. Y2H on ICD interactions' incorrectly referred to 'Extended Data Fig. 4d' instead of 'Extended Data Fig. 3d' on page 3. Finally, the section 'Text 4. Interaction network analysis' incorrectly referred to 'Fig. 1b and Extended Data Fig. 6' instead of 'Fig. 2b and Extended Data Fig. 7' on page 3. These errors have all been corrected in the Supplementary Information.

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

Role of jasmonate signaling in the regulation of plant responses to nutrient deficiency.

Phytohormones regulate plant growth and development by integrating various internal developmental cues with external environmental conditions. Jasmonic acid (JA) is an oxylipin-derived phytohormone that modulates the plasticity of plant responses against fluctuating environmental cues. An increasing number of studies have shown that it regulates a wide spectrum of plant physiological and biochemical processes, including reproductive development, primary root growth, root hair development, seed germination, senescence, regeneration, defense against biotic stress such as pathogen infection and herbivory, and mitigation of a number of abiotic stresses such as salinity, drought, high and low temperatures, wounding, excessive UV exposure, limited water availability, and metal(oid)-induced toxicity. Nutrient deficiency is an abiotic stress that adversely affects plant growth, development, and productivity, and JA also plays an important role in regulation of these processes under such conditions. In this review, we summarize recent advances relating to the role of JA and its methyl ester derivative (methyl jasmonate) in modulating responses to nutrient deficiency, to the impact of nutrient status on JA biosynthesis and signaling, and to the crosstalk of JA with other phytohormones in shaping plant growth and development under deficiencies of various mineral elements.

Protein targeting to starch 1, a functional protein of starch biosynthesis in wheat (Triticum aestivum L.).

KEY MESSAGE: TaPTST1, a wheat homolog of AtPTST1 containing CBM can interact with GBSSI and regulate starch metabolism in wheat endosperm. In cereal endosperm, native starch comprising amylose and amylopectin is synthesized by the coordinated activities of several pathway enzymes. Amylose in starch influences its physio-chemical properties resulting in several human health benefits. The Granule-Bound Starch Synthase I (GBSSI) is the most abundant starch-associated protein. GBSSI lacks dedicated Carbohydrate-binding module (CBM). Previously, Protein Targeting To Starch 1 (PTST1) was identified as a crucial protein for the localization of GBSSI to the starch granules in Arabidopsis. The function of its homologous protein in the wheat endosperm is not known. In this study, TaPTST1, an AtPTST1 homolog, containing a CBM and a coiled-coil domain was identified in wheat. Protein-coding nucleotide sequence of TaPTST1 from Indian wheat variety 'C 306' was cloned and characterized. Homology modelling and molecular docking suggested the potential interaction of TaPTST1 with glucans and GBSSI. The TaPTST1 expression was higher in wheat grain than the other tissues, suggesting a grain-specific function. In vitro binding assays demonstrated different binding affinities of TaPTST1 for native starch, amylose, and amylopectin. Furthermore, the immunoaffinity pull-down assay revealed that TaPTST1 directly interacts with GBSSI, and the interaction is mediated by a coiled-coil domain. The direct protein-protein interaction was further confirmed by bimolecular fluorescence complementation assay (BiFC) in planta. Based on our findings we postulate a functional role for TaPTST1 in starch metabolism by targeting GBSSI to starch granules in wheat endosperm.

Systems genomics approaches provide new insights into Arabidopsis thaliana root growth regulation under combinatorial mineral nutrient limitation.

The molecular mechanisms by which plants modulate their root growth rate (RGR) in response to nutrient deficiency are largely unknown. Using Arabidopsis thaliana accessions, we analyzed RGR variation under combinatorial mineral nutrient deficiencies involving phosphorus (P), iron (Fe), and zinc (Zn). While -P stimulated early RGR of most accessions, -Fe or -Zn reduced it. The combination of either -P-Fe or -P-Zn led to suppression of the growth inhibition exerted by -Fe or -Zn alone. Surprisingly, root growth responses of the reference accession Columbia (Col-0) were not representative of the species under -P nor -Zn. Using a systems approach that combines GWAS, network-based candidate identification, and reverse genetic screen, we identified new genes that regulate root growth in -P-Fe: VIM1, FH6, and VDAC3. Our findings provide a framework to systematically identifying favorable allelic variations to improve root growth, and to better understand how plants sense and respond to multiple environmental cues.

ELONGATED HYPOCOTYL5 Negatively Regulates DECREASE WAX BIOSYNTHESIS to Increase Survival during UV-B Stress.

Understanding how the distinct cell types of the shoot apical meristem (SAM) withstand ultraviolet radiation (UVR) stress can improve cultivation of plants in high-UVR environments. Here, we show that UV-B irradiation selectively kills epidermal and niche cells in the shoot apex. Plants harboring a mutation in DECREASE WAX BIOSYNTHESIS (DEWAX) are tolerant to UV-B. Our data show that DEWAX negatively regulates genes involved in anthocyanin biosynthesis. ELONGATED HYPOCOTYL5 (HY5) binds to the DEWAX promoter elements and represses its expression to promote the anthocyanin biosynthesis. The HY5-DEWAX regulatory network regulates anthocyanin content in Arabidopsis (Arabidopsis thaliana) and influences the survivability of plants under UV-B irradiation stress. Our cell sorting-based study of the epidermal cell layer transcriptome confirms that core UV-B stress signaling pathway genes are conserved and upregulated in response to UV-B irradiation of the SAM. Furthermore, we show that UV-B induces genes involved in shoot development and organ patterning. We propose that the HY5-DEWAX regulatory relationship is conserved; however, changes in the expression levels of these genes can determine anthocyanin content in planta and, hence, fitness under UV-B irradiation stress.

A genome-wide association study reveals cytokinin as a major component in the root defense responses against Ralstonia solanacearum.

Bacterial wilt caused by the soil-borne pathogen Ralstonia solancearum is economically devastating, with no effective methods to fight the disease. This pathogen invades plants through their roots and colonizes their xylem, clogging the vasculature and causing rapid wilting. Key to preventing colonization are the early defense responses triggered in the host's root upon infection, which remain mostly unknown. Here, we have taken advantage of a high-throughput in vitro infection system to screen natural variability associated with the root growth inhibition phenotype caused by R. solanacearum in Arabidopsis during the first hours of infection. To analyze the genetic determinants of this trait, we have performed a genome-wide association study, identifying allelic variation at several loci related to cytokinin metabolism, including genes responsible for biosynthesis and degradation of cytokinin. Further, our data clearly demonstrate that cytokinin signaling is induced early during the infection process and cytokinin contributes to immunity against R. solanacearum. This study highlights a new role for cytokinin in root immunity, paving the way for future research that will help in understanding the mechanisms underpinning root defenses.

Natural allelic variation of the AZI1 gene controls root growth under zinc-limiting condition.

Zinc is an essential micronutrient for all living organisms and is involved in a plethora of processes including growth and development, and immunity. However, it is unknown if there is a common genetic and molecular basis underlying multiple facets of zinc function. Here we used natural variation in Arabidopsis thaliana to study the role of zinc in regulating growth. We identify allelic variation of the systemic immunity gene AZI1 as a key for determining root growth responses to low zinc conditions. We further demonstrate that this gene is important for modulating primary root length depending on the zinc and defence status. Finally, we show that the interaction of the immunity signal azelaic acid and zinc level to regulate root growth is conserved in rice. This work demonstrates that there is a common genetic and molecular basis for multiple zinc dependent processes and that nutrient cues can determine the balance of growth and immune responses in plants.

Genetic control of root growth: from genes to networks.

BACKGROUND: Roots are essential organs for higher plants. They provide the plant with nutrients and water, anchor the plant in the soil, and can serve as energy storage organs. One remarkable feature of roots is that they are able to adjust their growth to changing environments. This adjustment is possible through mechanisms that modulate a diverse set of root traits such as growth rate, diameter, growth direction and lateral root formation. The basis of these traits and their modulation are at the cellular level, where a multitude of genes and gene networks precisely regulate development in time and space and tune it to environmental conditions. SCOPE: This review first describes the root system and then presents fundamental work that has shed light on the basic regulatory principles of root growth and development. It then considers emerging complexities and how they have been addressed using systems-biology approaches, and then describes and argues for a systems-genetics approach. For reasons of simplicity and conciseness, this review is mostly limited to work from the model plant Arabidopsis thaliana, in which much of the research in root growth regulation at the molecular level has been conducted. CONCLUSIONS: While forward genetic approaches have identified key regulators and genetic pathways, systems-biology approaches have been successful in shedding light on complex biological processes, for instance molecular mechanisms involving the quantitative interaction of several molecular components, or the interaction of large numbers of genes. However, there are significant limitations in many of these methods for capturing dynamic processes, as well as relating these processes to genotypic and phenotypic variation. The emerging field of systems genetics promises to overcome some of these limitations by linking genotypes to complex phenotypic and molecular data using approaches from different fields, such as genetics, genomics, systems biology and phenomics.

Underground tuning: quantitative regulation of root growth.

Plants display a high degree of phenotypic plasticity that allows them to tune their form and function to changing environments. The plant root system has evolved mechanisms to anchor the plant and to efficiently explore soils to forage for soil resources. Key to this is an enormous capacity for plasticity of multiple traits that shape the distribution of roots in the soil. Such root system architecture-related traits are determined by root growth rates, root growth direction, and root branching. In this review, we describe how the root system is constituted, and which mechanisms, pathways, and genes mainly regulate plasticity of the root system in response to environmental variation.

SOD-GIF-FIT module controls plant organ size and iron uptake.

Plant organ growth is controlled by various internal and external cues. However, the underlying molecular mechanisms that coordinate plant organ growth and nutrient homeostasis remain largely unknown. Recently, Zheng et al. identified the key regulators SOD7 (suppressor of da1-1) and GRF-INTERACTING FACTOR1 (GIF1) that control organ size and iron uptake in arabidopsis (Arabidopsis thaliana).

Paclobutrazol improves surface water use efficiency by regulating allometric trait behavior in maize.

Paclobutrazol (PBZ) role in drought management of maize is least understood. In maize, root traits are linked with surface water management. Over three years, early and terminal deficit irrigation (EDI and TDI) with or without PBZ were imposed on DKC-9144 and PG-2475 maize varieties. Several allometric parameters viz. stem height, stem diameter, leaf area and root traits along with physiological processes were measured. Implication of these parameters in the management of soil surface irrigation in terms of water use efficiency (WUE) was demonstrated in maize. Increased number of lateral roots and root number density in DKC-9144 provided more surface area for water absorption for better management of EDI. Root growth rates showed a similar pattern with root length, root surface areas, and root numbers in EDI. Elevated expressions of ZmRTCL, ZmRTCS and ZmARF34 in EDI and EDI plus PBZ were associated with seminal roots and root laterals initiation. Under TDI alone or in combination with PBZ, root lengths (BRL, CRL, SRL) and root surface areas varied in DKC-9144 and PG-2475 over control. Furthermore, correlation analysis showed that decrease in WUE under TDI was significantly associated with a reduction in stem thickness and leaf surface area. For WUE_N in TDI and PBZ plus TDI, structural equation modelling proposed, brace root surface area (BRSA_N) as a positive contributor, while a negative contributor was seminal root surface area (SRSA_N). Present study explained the importance of specific root traits and their association with other allometric parameters for improving WUE in DKC-9144 variety of maize and the crop in general.

The receptor kinase SRF3 coordinates iron-level and flagellin dependent defense and growth responses in plants.

Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remains unknown. Here we report a receptor kinase SRF3, with a role in coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthases. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domains which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

Heterologous expression and functional characterization of a Physcomitrella Pseudo response regulator homolog, PpPRR2, in Arabidopsis.

Physcomitrella patens has four homologs of the pseudo-response regulator involved in the circadian clock mechanism in seed plants. To gain insight into their function, Arabidopsis transgenic lines misexpressing PpPRR2 were constructed. Phenotypic analysis of the transformants with reference to clock-related gene expression and photoperiodic responses revealed that heterologous expression of the moss PpPRR2 gene modifies the intrinsic mechanism underlying the circadian clock in Arabidopsis, suggesting that PpPRR2 serves as a clock component in P. patens.

Functional characterization of CCA1/LHY homolog genes, PpCCA1a and PpCCA1b, in the moss Physcomitrella patens.

The evolution of circadian clocks in land plants is not understood, because circadian rhythms have received little attention in plants other than angiosperms. We have characterized two genes, PpCCA1a and PpCCA1b, homologs of the Arabidopsis thaliana clock genes CCA1/LHY, from the moss Physcomitrella patens. PpCCA1a and PpCCA1b, together with angiosperm CCA1/LHY homologs, belong to the clock-associated single-myb gene family of green plants (including green algae and land plants). The accumulation of PpCCA1a and PpCCA1b mRNA showed rhythms with a period of approximately 1 day, phased as are those of angiosperm homologs, under 24 h light/dark cycles or in continuous dark. However, in marked contrast to angiosperm homologs, both genes showed arrhythmic profiles in continuous light. The timing of the PpCCA1b peak is determined by the time of the last light to dark transition, suggesting that the arrhythmicity in continuous light is due to dysfunction of the core clock. We generated single and double disruptants for PpCCA1a and PpCCA1b, and found that the double disruptants showed: (i) short periodicity and damped amplitude in the PpCCA1b rhythm, (ii) similar changes in the rhythmically expressed genes PpSIG5 and PpPRRa, and (iii) de-repression of PpCCA1b transcription levels, indicating negative feedback regulation. These observations indicate that the two genes are not merely structural homologs but also functional counterparts of CCA1/LHY. Together, our results illustrate similarities as well as divergence of the clock machineries between P. patens and A. thaliana, two distantly placed species in land plant phylogeny.

Photoperiod-dependent regulation of cell growth by PpCCA1a and PpCCA1b genes encoding single-myb clock proteins in the moss Physcomitrella patens.

The PpCCA1a and PpCCA1b genes of the moss Physcomitrella patens are functional homologs of the Arabidopsis thaliana circadian clock genes CCA1/LHY. We made use of disruptant lines for PpCCA1a and/or PpCCA1b to elucidate the physiological significance of these genes in the growth of moss protonemal tissue under alternating day/night cycles. Protonemal cells of the double disruptant line, carrying neither of the two genes, grew faster than those of the wild-type plant (WT) in long days (LD), whereas no difference in the growth rate was detected between them in short days (SD). The double disruptant line also showed day length-dependent phenotypic changes in the PpCCA1b promoter activity: the diurnal profile of bioluminescence from the P(CCA1b)::LUC+ reporter strain was more significantly affected in LD than in SD. These observations are the first demonstration of a physiological function of the circadian clock in non-angiosperm land plants, and are consistent with recent findings that the clock controls hypocotyl elongation of A. thaliana in a photoperiod-dependent manner.

Automated High-Throughput Root Phenotyping of Arabidopsis thaliana Under Nutrient Deficiency Conditions.

The central question of genetics is how a genotype determines the phenotype of an organism. Genetic mapping approaches are a key for finding answers to this question. In particular, genome-wide association (GWA) studies have been rapidly adopted to study the architecture of complex quantitative traits. This was only possible due to the improvement of high-throughput and low-cost phenotyping methodologies. In this chapter we provide a detailed protocol for obtaining root trait data from the model species Arabidopsis thaliana using the semiautomated, high-throughput phenotyping pipeline BRAT (Busch-lab Root Analysis Toolchain) for early root growth under the stress condition of iron deficiency. Extracted root trait data can be directly used to perform GWA mapping using the freely accessible web application GWAPP to identify marker polymorphisms associated with the phenotype of interest.