PHB3 Regulates Lateral Root Primordia Formation via NO-mediated Degradation of AUX/IAAs

We previously showed that PHB3 regulates auxin-stimulated lateral root (LR) formation; however, the underlying molecular mechanism is unknown. Here, we demonstrate that PHB3 regulates LR development mainly through influencing lateral root primordia (LRP) initiation via affecting nitric oxide (NO) accumulation. The reduced LRP in phb3 was largely rescued by exogenous NO donor SNAP treatment. The decreased NO accumulation in phb3 caused a lower expression of GATA23 and LBD16 through inhibiting the degradation of IAA14/28. Overexpression of either GATA23 or LBD16 in phb3 mutant background recovered the reduced LRP number phenotype. These results indicate that PHB3 regulates LRP initiation via NO-mediated auxin signaling through regulating the degradation of IAA14/28.

B1L regulates lateral root development by exocytic vesicular trafficking-mediated polar auxin transport

Auxin and auxin-mediated signaling pathways involved in the regulation of lateral root development are well documented. Although exocytic vesicle trafficking plays an important role in PIN-auxin-efflux carrier recycling, and polar auxin transport during lateral root formation, however, the mechanistic details of these processes are not well understood. Here, we demonstrate an essential regulatory mechanism of B1L that interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation. B1L is highly expressed in Arabidopsis roots, and genetic and cellular analyses have revealed that B1L is mainly involved in lateral root primordia initiation. Furthermore, DR5::GUS expression analyses revealed that auxin levels were higher in lateral root primordia of the b1l mutant than in the wild-type. Exogenous auxin treatment confirmed that the lateral root phenotype correlated closely with auxin levels. Additionally, auxin transport-inhibitory treatment indicated that B1L regulates auxin efflux. Consistently, b1l mutants exhibited higher levels of auxin efflux carriers PIN1-GFP and PIN3-GFP in lateral root primordia. Moreover, B1L interacts with the exocyst and functions in recycling PIN2-GFP. Finally, the b1l-1/exo70b1-1 double-mutant exhibited a significant increase in the number of lateral roots compared to the wildtype, b1l-1, and exo70b1-1. Collectively, this study improves our understanding of the highly sophisticated processes involved in exocytic vesicular trafficking-mediated polar auxin transport and lateral root initiation in plants.

Polyamine Metabolism Is Involved in the Direct Regeneration of Shoots from Arabidopsis Lateral Root Primordia

Plants can be regenerated from various explants/tissues via de novo shoot meristem formation. Most of these regeneration pathways are indirect and involve callus formation. Besides plant hormones, the role of polyamines (PAs) has been implicated in these processes. Interestingly, the lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems by exogenous cytokinin application. In this system, no callus formation takes place. We report that the level of PAs, especially that of spermidine (Spd), increased during meristem conversion and the application of exogenous Spd improved its efficiency. The high endogenous Spd level could be due to enhanced synthesis as indicated by the augmented relative expression of PA synthesis genes (AtADC1,2, AtSAMDC2,4, AtSPDS1,2) during the process. However, the effect of PAs on shoot meristem formation might also be dependent on their catabolism. The expression of Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) was shown to be specifically high during the process and its ectopic overexpression increased the LRP-to-shoot conversion efficiency. This was correlated with Spd accumulation in the roots and ROS accumulation in the converting LRPs. The potential ways how PAO5 may influence direct shoot organogenesis from Arabidopsis LRPs are discussed.

The Arabidopsis TTL3 protein interconnects brassinosteroid signalling and cytoskeleton during lateral root development

Lateral roots are essential components of the plant edaphic interface, contributing to water and nutrient uptake, biotic and abiotic interactions, stress survival, and plant anchorage. We have identified the TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 3 (TTL3) being related to lateral root emergence and later development. TTL3 interacts with microtubules and potentially interconnects cytoskeletal function with the brassinosteroid signalling pathway. Loss of function of TTL3 leads to a reduced number of emerged lateral roots due to delayed development of lateral root primordia. Lateral root growth of the ttl3 mutant is less sensitive to BR treatment. Timing and spatial distribution of TTL3 expression is consistent with its role in development of lateral root primordia before their emergence and subsequent development into lateral roots. TTL3 is a novel component of the root system morphogenesis regulatory network.

Flavonols modulate lateral root emergence by scavenging reactive oxygen species in Arabidopsis thaliana

Flavonoids are a class of specialized metabolites with subclasses including flavonols and anthocyanins, which have unique properties as antioxidants. Flavonoids modulate plant development, but whether and how they impact lateral root development is unclear. We examined potential roles for flavonols in this process using Arabidopsis thaliana mutants with defects in genes encoding key enzymes in flavonoid biosynthesis. We observed the tt4 and fls1 mutants, which produce no flavonols, have increased lateral root emergence. The tt4 root phenotype was reversed by genetic and chemical complementation. To more specifically define the flavonoids involved, we tested an array of flavonoid biosynthetic mutants, eliminating roles for anthocyanins and the flavonols quercetin and isorhamnetin in modulating root development. Instead, two tt7 mutant alleles, with defects in a branchpoint enzyme blocking quercetin biosynthesis, formed reduced numbers of lateral roots, and tt7-2 had elevated levels of kaempferol. Using a flavonol-specific dye, we observed that in the tt7-2 mutant, kaempferol accumulated within lateral root primordia at higher levels than wild-type. These data are consistent with kaempferol, or downstream derivatives, acting as a negative regulator of lateral root emergence. We examined ROS accumulation using ROS-responsive probes and found reduced fluorescence of a superoxide-selective probe within the primordia of tt7-2 compared to wild type, but not in the tt4 mutant, consistent with opposite effects of these mutants on lateral root emergence. These results support a model in which increased level of kaempferol in the lateral root primordia of tt7-2 reduces superoxide concentration and ROS-stimulated lateral root emergence.

Transcriptome profiling of laser-captured crown root primordia reveals new pathways activated during early stages of crown root formation in rice

Crown roots constitute the main part of the rice root system. Several key genes involved in crown root initiation and development have been identified by functional genomics approaches. Nevertheless, these approaches are impaired by functional redundancy and mutant lethality. To overcome these limitations, organ targeted transcriptome analysis can help to identify genes involved in crown root formation and early development. In this study, we generated an atlas of genes expressed in developing crown root primordia in comparison with adjacent stem cortical tissue at three different developmental stages before emergence, using laser capture microdissection. We identified 3975 genes differentially expressed in crown root primordia. About 30% of them were expressed at the three developmental stages, whereas 10.5%, 19.5% and 12.8% were specifically expressed at the early, intermediate and late stages, respectively. Sorting them by functional ontology highlighted an active transcriptional switch during the process of crown root primordia formation. Cross-analysis with other rice root development-related datasets revealed genes encoding transcription factors, chromatin remodeling factors, peptide growth factors, and cell wall remodeling enzymes that are likely to play a key role during crown root primordia formation. This atlas constitutes an open primary data resource for further studies on the regulation of crown root initiation and development.

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis

In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.

Overexpression of the manganese/cadmium transporter OsNRAMP5 reduces cadmium accumulation in rice grain

Rice is a major dietary source of the toxic metal cadmium (Cd), and reducing its accumulation in the grain is therefore important for food safety. We selected two cultivars with contrasting Cd accumulation and generated transgenic lines overexpressing OsNRAMP5, which encodes a major influx transporter for manganese (Mn) and Cd. We used two different promoters to control the expression, namely OsActin1 and maize Ubiquitin. Overexpression of OsNRAMP5 increased Cd and Mn uptake into the roots, but markedly decreased Cd accumulation in the shoots, whilst having a relatively small effect on Mn accumulation in the shoots. The overexpressed OsNRAMP5 protein was localized to the plasma membrane of all cell types in the root tips and lateral root primordia without polarity. Synchrotron X-ray fluorescence mapping showed that the overexpression lines accumulated more Cd in the root tips and lateral root primordia compared with the wild-type. When grown in three Cd-contaminated paddy soils, overexpression of OsNRAMP5 decreased concentration of Cd in the grain by 49–94% compared with the wild type. OsNRAMP5-overexpression plants had decreased Cd translocation from roots to shoots as a result of disruption of its radial transport into the stele for xylem loading, demonstrating the effect of transporter localization and polarity on ion homeostasis.

Genome-Wide High Resolution Expression Map and Functions of Key Cell Fate Determinants Reveal the Dynamics of Crown Root Development in Rice

ABSTRACTRice adventitious/crown roots developing from non-root tissues shape up the root architecture. Mechanisms underlying initiation and subsequent outgrowth of CR primordia (CRP) remain under explored. Here, we provide genome-wide dynamics of gene expression patterns and stage-specific transcriptional signatures at distinct developmental stages of CRP formation. Our analyses reveal that early regulated transcription of potential epigenetic modifiers, transcription factors and cell division regulators prime the initiation of CRP followed by progressive activation of auxin signaling modules ensure their outgrowth. In depth analysis of spatio-temporal expression patterns of key cell fate determinants and functional analyses of rice WUSCHEL RELATED HOMEOBOX10 (OsWOX10) and PLETHORA (OsPLT) genes reveal their unprecedented role in CRP development. Our study suggests that OsWOX10 activates OsERF3 and OsCRL1 expression during CRP initiation and OsPLTs expression to accomplish their outgrowth. Interestingly, OsPLT genes, when expressed in the transcriptional domain of root-borne lateral root primordia of Arabidopsis plt mutant, rescued their outgrowth demonstrating the conserved role of PLT genes in root primordia outgrowth irrespective of their developmental origin. Together, these findings unveil the molecular framework of cellular reprogramming during trans-differentiation of shoot tissue to root leading to culmination of robust root architecture in monocot species which got evolutionary diverged from dicots.