Cytokinin signaling promotes root hair growth by directly regulating RSL4 expression.

Root hairs are single-celled tubular structures produced from the epidermis, which play an essential role in water and nutrient uptake from the soil. Therefore, root hair formation and elongation are controlled not only by developmental programs but also by environmental factors, enabling plants to survive under fluctuating conditions. Phytohormones are key signals that link environmental cues to developmental programs; indeed, root hair elongation is known to be controlled by auxin and ethylene. Another phytohormone, cytokinin, also affects root hair growth, while whether cytokinin is actively involved in root hair growth and, if so, how it regulates the signaling pathway governing root hair development have remained unknown. In this study, we show that the two-component system of cytokinin, which involves the B-type response regulators ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12, promotes the elongation process of root hairs. They directly up-regulate ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) encoding a basic helix-loop-helix (bHLH) transcription factor that plays a central role in root hair growth, whereas the ARR1/12-RSL4 pathway does not crosstalk with auxin or ethylene signaling. These results indicate that cytokinin signaling constitutes another input onto the regulatory module governed by RSL4, making it possible to fine-tune root hair growth in changing environments.

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light.

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.

Trihelix transcription factors GTL1 and DF1 prevent aberrant root hair formation in an excess nutrient condition.

Root hair growth is tuned in response to the environment surrounding plants. While most previous studies focused on the enhancement of root hair growth during nutrient starvation, few studies investigated the root hair response in the presence of excess nutrients. We report that the post-embryonic growth of wild-type Arabidopsis plants is strongly suppressed with increasing nutrient availability, particularly in the case of root hair growth. We further used gene expression profiling to analyze how excess nutrient availability affects root hair growth, and found that RHD6 subfamily genes, which are positive regulators of root hair growth, are downregulated in this condition. However, defects in GTL1 and DF1, which are negative regulators of root hair growth, cause frail and swollen root hairs to form when excess nutrients are supplied. Additionally, we observed that the RHD6 subfamily genes are mis-expressed in gtl1-1 df1-1. Furthermore, overexpression of RSL4, an RHD6 subfamily gene, induces swollen root hairs in the face of a nutrient overload, while mutation of RSL4 in gtl1-1 df1-1 restore root hair swelling phenotype. In conclusion, our data suggest that GTL1 and DF1 prevent unnecessary root hair formation by repressing RSL4 under excess nutrient conditions.

Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity.

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.

AT-Hook Transcription Factors Restrict Petiole Growth by Antagonizing PIFs.

Upon detecting abiotic or biotic stress, plants generally reduce their growth, enabling resources to be conserved and diverted to stress response mechanisms. In Arabidopsis thaliana, the AT-hook motif nuclear-localized (AHL) transcription factor family has been implicated in restricting rosette growth in response to stress. However, the mechanism by which AHLs repress growth in rosettes is unknown. In this study, we establish that SUPPRESSOR OF PHYTOCHROME B4-#3 (SOB3) and other AHLs restrict petiole elongation by antagonizing the growth-promoting PHYTOCHROME-INTERACTING FACTORs (PIFs). Our data show that high levels of SOB3 expression lead to a short-petiole phenotype similar to that conferred by removal of PIF4. Conversely, the dominant-negative sob3-6 mutant has long petioles, a phenotype which is PIF-dependent. We further show that AHLs repress the expression of many PIF-activated genes, several of which are involved in hormone-mediated promotion of growth. Additionally, a subset of PIF-activated, AHL-repressed genes are directly bound by both SOB3 and PIFs. Finally, SOB3 reduces binding of PIF4 to shared target loci. Collectively, our results demonstrate that AHLs repress petiole growth by antagonizing PIF-mediated transcriptional activation of genes associated with growth and hormone pathways. By elucidating a mechanism via which the stress-responsive AHL transcription factor family influences growth in petioles, this study identifies a key step in the gene regulatory network controlling leaf growth in response to the environment.

A gene regulatory network for root hair development.

Root hairs play important roles for the acquisition of nutrients, microbe interaction and plant anchorage. In addition, root hairs provide an excellent model system to study cell patterning, differentiation and growth. Arabidopsis root hairs have been thoroughly studied to understand how plants regulate cell fate and growth in response to environmental signals. Accumulating evidence suggests that a multi-layered gene regulatory network is the molecular secret to enable the flexible and adequate response to multiple signals. In this review, we describe the key transcriptional regulators controlling cell fate and/or cell growth of root hairs. We also discuss how plants integrate phytohormonal and environmental signals, such as auxin, ethylene and phosphate availability, and modulate the level of these transcriptional regulators to tune root hair development.

A Gene Regulatory Network for Cellular Reprogramming in Plant Regeneration.

Wounding triggers organ regeneration in many plant species, and application of plant hormones, such as auxin and cytokinin, enhances their regenerative capacities in tissue culture. Recent studies have identified several key players mediating wound- and/or plant hormone-induced cellular reprogramming, but the global architecture of gene regulatory relationships underlying plant cellular reprogramming is still far from clear. In this study, we uncovered a gene regulatory network (GRN) associated with plant cellular reprogramming by using an enhanced yeast one-hybrid (eY1H) screen systematically to identify regulatory relationships between 252 transcription factors (TFs) and 48 promoters. Our network analyses suggest that wound- and/or hormone-invoked signals exhibit extensive cross-talk and regulate many common reprogramming-associated genes via multilayered regulatory cascades. Our data suggest that PLETHORA 3 (PLT3), ENHANCER OF SHOOT REGENERATION 1 (ESR1) and HEAT SHOCK FACTOR B 1 (HSFB1) act as critical nodes that have many overlapping targets and potentially connect upstream stimuli to downstream developmental decisions. Interestingly, a set of wound-inducible APETALA 2/ETHYLENE RESPONSE FACTORs (AP2/ERFs) appear to regulate these key genes, which, in turn, form feed-forward cascades that control downstream targets associated with callus formation and organ regeneration. In addition, we found another regulatory pathway, mediated by LATERAL ORGAN BOUNDARY/ASYMMETRIC LEAVES 2 (LOB/AS2) TFs, which probably plays a distinct but partially overlapping role alongside the AP2/ERFs in the putative gene regulatory cascades. Taken together, our findings provide the first global picture of the GRN governing plant cell reprogramming, which will serve as a valuable resource for future studies.

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis.

How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors.

ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator.

Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2 Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis.

Molecular networks orchestrating plant cell growth.

Plant cell growth can broadly be categorized into either diffuse or tip growth. Here we compare gene regulatory networks (GRNs) controlling growth of hypocotyls and root hairs as examples for diffuse and tip growth, respectively. Accumulating evidence shows that GRNs in both cell types are multi-layered in structure and fine-tuned by transcriptional and post-translational mechanisms. We discuss how these GRNs regulate the expression of proteins controlling cell wall remodeling or other growth regulatory processes. Finally, we highlight how specific regulators within GRNs adjust plant cell growth in response to variable environmental conditions.

PRC2 represses dedifferentiation of mature somatic cells in Arabidopsis.

Plant somatic cells are generally acknowledged to retain totipotency, the potential to develop into any cell type within an organism. This astonishing plasticity may contribute to a high regenerative capacity on severe damage, but how plants control this potential during normal post-embryonic development remains largely unknown(1,2). Here we show that POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a chromatin regulator that maintains gene repression through histone modification, prevents dedifferentiation of mature somatic cells in Arabidopsis thaliana roots. Loss-of-function mutants in PRC2 subunits initially develop unicellular root hairs indistinguishable from those in wild type but fail to retain the differentiated state, ultimately resulting in the generation of an unorganized cell mass and somatic embryos from a single root hair. Strikingly, mutant root hairs complete the normal endoreduplication programme, increasing their nuclear ploidy, but subsequently reinitiate mitotic division coupled with successive DNA replication. Our data show that the WOUND INDUCED DEDIFFERENTIATION3 (WIND3) and LEAFY COTYLEDON2 (LEC2) genes are among the PRC2 targets involved in this reprogramming, as their ectopic overexpression partly phenocopies the dedifferentiation phenotype of PRC2 mutants. These findings unveil the pivotal role of PRC2-mediated gene repression in preventing unscheduled reprogramming of fully differentiated plant cells.

Interaction between chaperone and protease functions of LON2, and autophagy during the functional transition of peroxisomes.

Functional transition of glyoxysomes to leaf peroxisomes is observed in greening cotyledons. Glyoxysomal proteins are rapidly degraded and leaf-peroxisomal proteins are transported into peroxisomes after cotyledons are exposed to light, but the molecular mechanisms underlying these processes remain unclear. We recently discovered that two degradation pathways are involved in the functional transition of peroxisomes using Arabidopsis thaliana. Lon protease 2 (LON2) is responsible for the degradation of glyoxysomal proteins inside peroxisomes, and, in parallel, autophagy eliminates damaged or obsolete peroxisomes. A double mutant defective in both the LON2- and autophagy-dependent degradation pathways accumulated glyoxysomal proteins after the cotyledons became green. Our study also demonstrated that the LON2- and autophagy-dependent pathways are interdependent, with the chaperone function of LON2 suppressing autophagic peroxisome degradation. Moreover, the peptidase domain of LON2 interferes with the suppression of autophagy, indicating that autophagy is regulated by intramolecular modulation between the proteolysis and chaperone functions of LON2.

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality.

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.

Organ-specific quality control of plant peroxisomes is mediated by autophagy.

Peroxisomes are essential organelles that are characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG)-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5-knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles for degradation by autophagy. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves and for proper plant development under natural growth conditions.

Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.