Dual regulations of cell cycle regulator DPa by auxin in Arabidopsis root distal stem cell maintenance.

The plant hormone auxin plays a key role to maintain root stem cell identity which is essential for root development. However, the molecular mechanism by which auxin regulates root distal stem cell (DSC) identity is not well understood. In this study, we revealed that the cell cycle factor DPa is a vital regulator in the maintenance of root DSC identity through multiple auxin signaling cascades. On the one hand, auxin positively regulates the transcription of DPa via AUXIN RESPONSE FACTOR 7 and ARF19. On the other hand, auxin enhances the protein stability of DPa through MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3)/MPK6-mediated phosphorylation. Consistently, mutation of the identified three threonine residues (Thr10, Thr25, and Thr227) of DPa to nonphosphorylated form alanine (DPa3A) highly decreased the phosphorylation level of DPa, which decreased its protein stability and affected the maintenance of root DSC identity. Taken together, this study provides insight into the molecular mechanism of how auxin regulates root distal stem cell identity through the dual regulations of DPa at both transcriptional and posttranslational levels.

Correction: PRH1 mediates ARF7-LBD dependent auxin signaling to regulate lateral root development in Arabidopsis thaliana.

[This corrects the article DOI: 10.1371/journal.pgen.1008044.].

Non-canonical AUX/IAA protein IAA33 competes with canonical AUX/IAA repressor IAA5 to negatively regulate auxin signaling.

The phytohormone auxin controls plant growth and development via TIR1-dependent protein degradation of canonical AUX/IAA proteins, which normally repress the activity of auxin response transcription factors (ARFs). IAA33 is a non-canonical AUX/IAA protein lacking a TIR1-binding domain, and its role in auxin signaling and plant development is not well understood. Here, we show that IAA33 maintains root distal stem cell identity and negatively regulates auxin signaling by interacting with ARF10 and ARF16. IAA33 competes with the canonical AUX/IAA repressor IAA5 for binding to ARF10/16 to protect them from IAA5-mediated inhibition. In contrast to auxin-dependent degradation of canonical AUX/IAA proteins, auxin stabilizes IAA33 protein via MITOGEN-ACTIVATED PROTEIN KINASE 14 (MPK14) and does not affect IAA33 gene expression. Taken together, this study provides insight into the molecular functions of non-canonical AUX/IAA proteins in auxin signaling transduction.

Correction: Local Transcriptional Control of YUCCA Regulates Auxin Promoted Root-Growth Inhibition in Response to Aluminium Stress in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pgen.1006360.].

MYB2 and MYB108 regulate lateral root development by interacting with LBD29 in Arabidopsis thaliana.

AUXIN RESPONSE FACTOR 7 (ARF7)-mediated auxin signaling plays a key role in lateral root (LR) development by regulating downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor genes, including LBD16, LBD18, and LBD29. LBD proteins are believed to regulate the transcription of downstream genes as homodimers or heterodimers. However, whether LBD29 forms dimers with other proteins to regulate LR development remains unknown. Here, we determined that the Arabidopsis thaliana (L.) Heynh. MYB transcription factors MYB2 and MYB108 interact with LBD29 and regulate auxin-induced LR development. Both MYB2 and MYB108 were induced by auxin in an ARF7-dependent manner. Disruption of MYB2 by fusion with an SRDX domain severely affected auxin-induced LR formation and the ability of LBD29 to induce LR development. By contrast, overexpression of MYB2 or MYB108 resulted in greater LR numbers, except in the lbd29 mutant background. These findings underscore the interdependence and importance of MYB2, MYB108, and LBD29 in regulating LR development. In addition, MYB2-LBD29 and MYB108-LBD29 complexes promoted the expression of CUTICLE DESTRUCTING FACTOR 1 (CDEF1), a member of the GDSL (Gly-Asp-Ser-Leu) lipase/esterase family involved in LR development. In summary, this study identified MYB2-LBD29 and MYB108-LBD29 regulatory modules that act downstream of ARF7 and intricately control auxin-mediated LR development.

MPK3/6-induced degradation of ARR1/10/12 promotes salt tolerance in Arabidopsis.

Cytokinins are phytohormones that regulate plant development, growth, and responses to stress. In particular, cytokinin has been reported to negatively regulate plant adaptation to high salinity; however, the molecular mechanisms that counteract cytokinin signaling and enable salt tolerance are not fully understood. Here, we provide evidence that salt stress induces the degradation of the cytokinin signaling components Arabidopsis (Arabidopisis thaliana) response regulator 1 (ARR1), ARR10 and ARR12. Furthermore, the stress-activated mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ARR1/10/12 to promote their degradation in response to salt stress. As expected, salt tolerance is decreased in the mpk3/6 double mutant, but enhanced upon ectopic MPK3/MPK6 activation in an MKK5DD line. Importantly, salt hypersensitivity phenotypes of the mpk3/6 line were significantly alleviated by mutation of ARR1/12. The above results indicate that MPK3/6 enhance salt tolerance in part via their negative regulation of ARR1/10/12 protein stability. Thus, our work reveals a new molecular mechanism underlying salt-induced stress adaptation and the inhibition of plant growth, via enhanced degradation of cytokinin signaling components.

PRH1 mediates ARF7-LBD dependent auxin signaling to regulate lateral root development in Arabidopsis thaliana.

The development of lateral roots in Arabidopsis thaliana is strongly dependent on signaling directed by the AUXIN RESPONSE FACTOR7 (ARF7), which in turn activates LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors (LBD16, LBD18 and LBD29). Here, the product of PRH1, a PR-1 homolog annotated previously as encoding a pathogen-responsive protein, was identified as a target of ARF7-mediated auxin signaling and also as participating in the development of lateral roots. PRH1 was shown to be strongly induced by auxin treatment, and plants lacking a functional copy of PRH1 formed fewer lateral roots. The transcription of PRH1 was controlled by the binding of both ARF7 and LBDs to its promoter region.

Cell-type action specificity of auxin on Arabidopsis root growth.

The plant hormone auxin plays a critical role in root growth and development; however, the contributions or specific roles of cell-type auxin signals in root growth and development are not well understood. Here, we mapped tissue and cell types that are important for auxin-mediated root growth and development by manipulating the local response and synthesis of auxin. Repressing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele strongly inhibited root growth, with the largest effect observed in the endodermis. Enhancing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele also caused reduced root growth, albeit to a lesser extent. Moreover, we established that root growth was inhibited by enhancement of auxin synthesis in specific cell types of the epidermis, cortex and endodermis, whereas increased auxin synthesis in the pericycle and stele had only minor effects on root growth. Our study thus establishes an association between cellular identity and cell type-specific auxin signaling that guides root growth and development.

Serratia marcescens PLR enhances lateral root formation through supplying PLR-derived auxin and enhancing auxin biosynthesis in Arabidopsis.

Plant growth promoting rhizobacteria (PGPR) refer to bacteria that colonize the rhizosphere and contribute to plant growth or stress tolerance. To further understand the molecular mechanism by which PGPR exhibit symbiosis with plants, we performed a high-throughput single colony screening from the rhizosphere, and uncovered a bacterium (named promoting lateral root, PLR) that significantly promotes Arabidopsis lateral root formation. By 16S rDNA sequencing, PLR was identified as a novel sub-species of Serratia marcescens. RNA-seq analysis of Arabidopsis integrated with phenotypic verification of auxin signalling mutants demonstrated that the promoting effect of PLR on lateral root formation is dependent on auxin signalling. Furthermore, PLR enhanced tryptophan-dependent indole-3-acetic acid (IAA) synthesis by inducing multiple auxin biosynthesis genes in Arabidopsis. Genome-wide sequencing of PLR integrated with the identification of IAA and its precursors in PLR exudates showed that tryptophan treatment significantly enhanced the ability of PLR to produce IAA and its precursors. Interestingly, PLR induced the expression of multiple nutrient (N, P, K, S) transporter genes in Arabidopsis in an auxin-independent manner. This study provides evidence of how PLR enhances plant growth through fine-tuning auxin biosynthesis and signalling in Arabidopsis, implying a potential application of PLR in crop yield improvement through accelerating root development.

CO2 is a key constituent of the plant growth-promoting volatiles generated by bacteria in a sealed system.

KEY MESSAGE: Plant growth is greatly inhibited in tightly sealed Petri dishes for lack of CO2. Bacteria which co-cultured with plant can produce CO2 to promote plant growth in sealed systems. Bacteria produce a wide variety of volatiles, some of which can support and others can damage plant growth. It is a controversial issue whether CO2 or other bacterial volatile compounds promote plant growth in sealed systems. CO2 is critical for photosynthesis. Here, we show that CO2 is a key constituent of the plant growth-promoting volatiles generated by bacteria in a sealed system. We revealed that the growth of Arabidopsis seedlings in an airtight container was retarded due to insufficient supply of the CO2. When either CO2 was introduced into the container, or the seedlings were co-cultured along with certain bacterial species, the plants' growth was restored. CONCLUSION: The benefit of co-culturing was largely due to the CO2 generated by respiration of the bacteria.

Brassinosteroids regulate root growth by controlling reactive oxygen species homeostasis and dual effect on ethylene synthesis in Arabidopsis.

The brassinosteroids (BRs) represent a class of phytohormones, which regulate numerous aspects of growth and development. Here, a det2-9 mutant defective in BR synthesis was identified from an EMS mutant screening for defects in root length, and was used to investigate the role of BR in root development in Arabidopsis. The det2-9 mutant displays a short-root phenotype, which is result from the reduced cell number in root meristem and decreased cell size in root maturation zone. Ethylene synthesis is highly increased in the det2-9 mutant compared with the wild type, resulting in the hyper-accumulation of ethylene and the consequent inhibition of root growth. The short-root phenotype of det2-9 was partially recovered in the det2-9/acs9 double mutant and det2-9/ein3/eil1-1 triple mutant which have defects either in ethylene synthesis or ethylene signaling, respectively. Exogenous application of BR showed that BRs either positively or negatively regulate ethylene biosynthesis in a concentration-dependent manner. Different from the BR induced ethylene biosynthesis through stabilizing ACSs stability, we found that the BR signaling transcription factors BES1 and BZR1 directly interacted with the promoters of ACS7, ACS9 and ACS11 to repress their expression, indicating a native regulation mechanism under physiological levels of BR. In addition, the det2-9 mutant displayed over accumulated superoxide anions (O2-) compared with the wild-type control, and the increased O2- level was shown to contribute to the inhibition of root growth. The BR-modulated control over the accumulation of O2- acted via the peroxidase pathway rather than via the NADPH oxidase pathway. This study reveals an important mechanism by which the hormone cross-regulation between BRs and ethylene or/and ROS is involved in controlling root growth and development in Arabidopsis.

SIZ1 negatively regulates aluminum resistance by mediating the STOP1-ALMT1 pathway in Arabidopsis.

Sensitive to proton rhizotoxicity 1 (STOP1) functions as a crucial regulator of root growth during aluminum (Al) stress. However, how this transcription factor is regulated by Al stress to affect downstream genes expression is not well understood. To explore the underlying mechanisms of the function and regulation of STOP1, we employed a yeast two hybrid screen to identify STOP1-interacting proteins. The SUMO E3 ligase SIZ1, was found to interact with STOP1 and mainly facilitate its SUMO modification at K40 and K212 residues. Simultaneous introduction of K40R and K212R substitutions in STOP1 enhances its transactivation activity to upregulate the expression of aluminum-activated malate transporter 1 (ALMT1) via increasing the association with mediator 16 (MED16) transcriptional co-activator. Loss of function of SIZ1 causes highly increased expression of ALMT1, thus enhancing Al-induced malate exudation and Al tolerance. Also, we found that the protein level of SIZ1 is reduced in response to Al stress. Genetic evidence demonstrates that STOP1/ALMT1 is epistatic to SIZ1 in regulating root growth response to Al stress. This study suggests a mechanism about how the SIZ1-STOP1-ALMT1 signaling module is involved in root growth response to Al stress.

The pre-mRNA splicing factor RDM16 regulates root stem cell maintenance in Arabidopsis.

Pre-mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre-mRNA splicing (rdm16-4). The rdm16-4 mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem. The PLETHORA1 (PLT1) and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16-4 mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap of rdm16-4 contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS (ARR1, ARR2, and ARR11). Furthermore, expression of the full-length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short-root phenotype of rdm16-4. This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.

Neural defects caused by total and Wnt1-Cre mediated ablation of p120ctn in mice.

BACKGROUND: p120 catenin (p120ctn) is an important component in the cadherin-catenin cell adhesion complex because it stabilizes cadherin-mediated intercellular junctions. Outside these junctions, p120ctn is actively involved in the regulation of small GTPases of the Rho family, in actomyosin dynamics and in transcription regulation. We and others reported that loss of p120ctn in mouse embryos results in an embryonic lethal phenotype, but the exact developmental role of p120ctn during brain formation has not been reported. RESULTS: We combined floxed p120ctn mice with Del-Cre or Wnt1-Cre mice to deplete p120ctn from either all cells or specific brain and neural crest cells. Complete loss of p120ctn in mid-gestation embryos resulted in an aberrant morphology, including growth retardation, failure to switch from lordotic to fetal posture, and defective neural tube formation and neurogenesis. By expressing a wild-type p120ctn from the ROSA26 locus in p120ctn-null mouse embryonic stem cells, we could partially rescue neurogenesis. To further investigate the developmental role of p120ctn in neural tube formation, we generated conditional p120ctnfl/fl;Wnt1Cre knockout mice. p120ctn deletion in Wnt1-expressing cells resulted in neural tube closure defects (NTDs) and craniofacial abnormalities. These defects could not be correlated with misregulation of brain marker genes or cell proliferation. In contrast, we found that p120ctn is required for proper expression of the cell adhesion components N-cadherin, E-cadherin and ß-catenin, and of actin-binding proteins cortactin and Shroom3 at the apical side of neural folds. This region is of critical importance for closure of neural folds. Surprisingly, the lateral side of mutant neural folds showed loss of p120ctn, but not of N-cadherin, ß-catenin or cortactin. CONCLUSIONS: These results indicate that p120ctn is required for neurogenesis and neurulation. Elimination of p120ctn in cells expressing Wnt1 affects neural tube closure by hampering correct formation of specific adhesion and actomyosin complexes at the apical side of neural folds. Collectively, our results demonstrate the crucial role of p120ctn during brain morphogenesis.

PIFs coordinate shade avoidance by inhibiting auxin repressor ARF18 and metabolic regulator QQS.

Shade avoidance syndrome (SAS) arises in densely growing plants that compete for light. In Arabidopsis thaliana, phytochrome interacting factor (PIF) proteins link the perception of shade to stem elongation via auxin production. Here, we report that PIFs inhibit the shade-induced expression of AUXIN RESPONSE FACTOR 18 (ARF18), and ARF18 represses auxin signaling. Therefore, PIF-mediated inhibition of ARF18 enhances auxin-dependent hypocotyl elongation in simulated shade. Furthermore, we show that both PIFs and ARF18 directly repress qua-quine starch (QQS), which controls the allocation of carbon and nitrogen. Shade-repressed QQS attenuates the conversion of starch to protein and thus reduced leaf area. Our results suggest that PIF-dependent gene regulation coordinates multiple SAS responses, including altered stem growth via ARF18, as well as altered leaf growth and metabolism via QQS.

Nanoplastics Promote Microcystin Synthesis and Release from Cyanobacterial Microcystis aeruginosa.

Although the fate of nanoplastics (<100 nm) in freshwater systems is increasingly well studied, much less is known about its potential threats to cyanobacterial blooms, the ultimate phenomenon of eutrophication occurrence worldwide. Previous studies have evaluated the consequences of nanoplastics increasing the membrane permeability of microbes, however, there is no direct evidence for interactions between nanoplastics and microcystin; intracellular hepatotoxins are produced by some genera of cyanobacteria. Here, we show that the amino-modified polystyrene nanoplastics (PS-NH2) promote microcystin synthesis and release from Microcystis aeruginosa, a dominant species causing cyanobacterial blooms, even without the change of coloration. We demonstrate that PS-NH2 inhibits photosystem II efficiency, reduces organic substance synthesis, and induces oxidative stress, enhancing the synthesis of microcystin. Furthermore, PS-NH2 promotes the extracellular release of microcystin from M. aeruginosa via transporter protein upregulation and impaired cell membrane integrity. Our findings propose that the presence of nanoplastics in freshwater ecosystems might enhance the threat of eutrophication to aquatic ecology and human health.

AtHB7/12 Regulate Root Growth in Response to Aluminum Stress.

Aluminum (Al) stress is a major limiting factor for plant growth and crop production in acid soils. At present, only a few transcription factors involved in the regulation of Al resistance have been characterized. Here, we used reversed genetic approach through phenotype analysis of overexpressors and mutants to demonstrate that AtHB7 and AtHB12, two HD-Zip I transcription factors, participate in Al resistance. In response to Al stress, AtHB7 and AtHB12 displayed different dynamic expression patterns. Although both AtHB7 and AtHB12 positively regulate root growth in the absence of Al stress, our results showed that AtHB7 antagonizes with AtHB12 to control root growth in response to Al stress. The athb7/12 double mutant displayed a wild-type phenotype under Al stress. Consistently, our physiological analysis showed that AtHB7 and AtHB12 oppositely regulate the capacity of cell wall to bind Al. Yeast two hybrid assays showed that AtHB7 and AtHB12 could form homo-dimers and hetero-dimers in vitro, suggesting the interaction between AtHB7 and AtHB12 in the regulation of root growth. The conclusion was that AtHB7 and AtHB12 oppositely regulate Al resistance by affecting Al accumulation in root cell wall.

Local Transcriptional Control of YUCCA Regulates Auxin Promoted Root-Growth Inhibition in Response to Aluminium Stress in Arabidopsis.

Auxin is necessary for the inhibition of root growth induced by aluminium (Al) stress, however the molecular mechanism controlling this is largely unknown. Here, we report that YUCCA (YUC), which encodes flavin monooxygenase-like proteins, regulates local auxin biosynthesis in the root apex transition zone (TZ) in response to Al stress. Al stress up-regulates YUC3/5/7/8/9 in the root-apex TZ, which we show results in the accumulation of auxin in the root-apex TZ and root-growth inhibition during the Al stress response. These Al-dependent changes in the regulation of YUCs in the root-apex TZ and YUC-regulated root growth inhibition are dependent on ethylene signalling. Increasing or disruption of ethylene signalling caused either enhanced or reduced up-regulation, respectively, of YUCs in root-apex TZ in response to Al stress. In addition, ethylene enhanced root growth inhibition under Al stress was strongly alleviated in yuc mutants or by co-treatment with yucasin, an inhibitor of YUC activity, suggesting a downstream role of YUCs in this process. Moreover, ethylene-insensitive 3 (EIN3) is involved into the direct regulation of YUC9 transcription in this process. Furthermore, we demonstrated that PHYTOCHROME INTERACTING FACTOR4 (PIF4) functions as a transcriptional activator for YUC5/8/9. PIF4 promotes Al-inhibited primary root growth by regulating the local expression of YUCs and auxin signal in the root-apex TZ. The Al-induced expression of PIF4 in root TZ acts downstream of ethylene signalling. Taken together, our results highlight a regulatory cascade for YUCs-regulated local auxin biosynthesis in the root-apex TZ mediating root growth inhibition in response to Al stress.

A P-Loop NTPase Regulates Quiescent Center Cell Division and Distal Stem Cell Identity through the Regulation of ROS Homeostasis in Arabidopsis Root.

Reactive oxygen species (ROS) are recognized as important regulators of cell division and differentiation. The Arabidopsis thaliana P-loop NTPase encoded by APP1 affects root stem cell niche identity through its control of local ROS homeostasis. The disruption of APP1 is accompanied by a reduction in ROS level, a rise in the rate of cell division in the quiescent center (QC) and the promotion of root distal stem cell (DSC) differentiation. Both the higher level of ROS induced in the app1 mutant by exposure to methyl viologen (MV), and treatment with hydrogen peroxide (H2O2) rescued the mutant phenotype, implying that both the increased rate of cell division in the QC and the enhancement in root DSC differentiation can be attributed to a low level of ROS. APP1 is expressed in the root apical meristem cell mitochondria, and its product is associated with ATP hydrolase activity. The key transcription factors, which are defining root distal stem niche, such as SCARECROW (SCR) and SHORT ROOT (SHR) are both significantly down-regulated at both the transcriptional and protein level in the app1 mutant, indicating that SHR and SCR are important downstream targets of APP1-regulated ROS signaling to control the identity of root QC and DSCs.