Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling.

SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.

Small signaling peptides mediate plant adaptions to abiotic environmental stress.

MAIN CONCLUSION: Peptide-receptor complexes activate distinct downstream regulatory networks to mediate plant adaptions to abiotic environmental stress. Plants are constantly exposed to various adverse environmental factors; thus they must adjust their growth accordingly. Plants recruit small secretory peptides to adapt to these detrimental environments. These small peptides, which are perceived by their corresponding receptors and/or co-receptors, act as local- or long-distance mobile signaling molecules to establish cell-to-cell regulatory networks, resulting in optimal cellular and physiological outputs. In this review, we highlight recent advances on the regulatory role of small peptides in plant abiotic responses and nutrients signaling.

Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking.

The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.

PIN-mediated polar auxin transport regulations in plant tropic responses.

Tropisms, growth responses to environmental stimuli such as light or gravity, are spectacular examples of adaptive plant development. The plant hormone auxin serves as a major coordinative signal. The PIN auxin exporters, through their dynamic polar subcellular localizations, redirect auxin fluxes in response to environmental stimuli and the resulting auxin gradients across organs underlie differential cell elongation and bending. In this review, we discuss recent advances concerning regulations of PIN polarity during tropisms, focusing on PIN phosphorylation and trafficking. We also cover how environmental cues regulate PIN actions during tropisms, as well as the crucial role of auxin feedback on PIN polarity during bending termination. Finally, the interactions between different tropisms are reviewed to understand plant adaptive growth in the natural environment.

Seedling developmental defects upon blocking CINNAMATE-4-HYDROXYLASE are caused by perturbations in auxin transport.

The phenylpropanoid pathway serves a central role in plant metabolism, providing numerous compounds involved in diverse physiological processes. Most carbon entering the pathway is incorporated into lignin. Although several phenylpropanoid pathway mutants show seedling growth arrest, the role for lignin in seedling growth and development is unexplored. We use complementary pharmacological and genetic approaches to block CINNAMATE-4-HYDROXYLASE (C4H) functionality in Arabidopsis seedlings and a set of molecular and biochemical techniques to investigate the underlying phenotypes. Blocking C4H resulted in reduced lateral rooting and increased adventitious rooting apically in the hypocotyl. These phenotypes coincided with an inhibition in AUX transport. The upstream accumulation in cis-cinnamic acid was found to be likely to cause polar AUX transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem-mediated AUX transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, AUX homeostasis. Our results show that the accumulation of bioactive intermediates and depletion in lignin jointly cause the aberrant phenotypes upon blocking C4H, and demonstrate that proper deposition of lignin is essential for the establishment of AUX distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development.

Genetic screen for factors mediating PIN polarization in gravistimulated Arabidopsis thaliana hypocotyls.

Gravitropism is an adaptive response that orients plant growth parallel to the gravity vector. Asymmetric distribution of the phytohormone auxin is a necessary prerequisite to the tropic bending both in roots and shoots. During hypocotyl gravitropic response, the PIN3 auxin transporter polarizes within gravity-sensing cells to redirect intercellular auxin fluxes. First gravity-induced PIN3 polarization to the bottom cell membranes leads to the auxin accumulation at the lower side of the organ, initiating bending and, later, auxin feedback-mediated repolarization restores symmetric auxin distribution to terminate bending. Here, we performed a forward genetic screen to identify regulators of both PIN3 polarization events during gravitropic response. We searched for mutants with defective PIN3 polarizations based on easy-to-score morphological outputs of decreased or increased gravity-induced hypocotyl bending. We identified the number of hypocotyl reduced bending (hrb) and hypocotyl hyperbending (hhb) mutants, revealing that reduced bending correlated typically with defective gravity-induced PIN3 relocation whereas all analyzed hhb mutants showed defects in the second, auxin-mediated PIN3 relocation. Next-generation sequencing-aided mutation mapping identified several candidate genes, including SCARECROW and ACTIN2, revealing roles of endodermis specification and actin cytoskeleton in the respective gravity- and auxin-induced PIN polarization events. The hypocotyl gravitropism screen thus promises to provide novel insights into mechanisms underlying cell polarity and plant adaptive development.

CLE9 peptide-induced stomatal closure is mediated by abscisic acid, hydrogen peroxide, and nitric oxide in Arabidopsis thaliana.

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss-of-function mutants were sensitivity to drought stress. CLE9-induced stomatal closure was impaired in abscisic acid (ABA)-deficient mutants, indicating that ABA is required for CLE9-medaited guard cell signalling. We further deciphered that two guard cell ABA-signalling components, OST1 and SLAC1, were responsible for CLE9-induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H2 O2 ) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase-deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA-dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.

Identification and characterization of the Populus trichocarpa CLE family.

BACKGROUND: The CLE (CLAVATA3/Endosperm Surrounding Region-related) gene family encodes small signaling peptides that are primarily involved in coordinating stem cell fate in different types of plant meristems. Their roles in vascular cambium have highlighted their potential function in wood formation. Apart from recent advances on identification and characterization of CLE genes, little is known about this gene family in a tree species. RESULTS: Fifty PtCLE genes were identified from the Populus trichocarpa genome and were classified into four major groups based on sequence similarity. Analysis of the genomic organization of PtCLE genes indicates that genome duplication, as well as the diversity in the CLE motif, have contributed to the expansion of CLE gene family in poplar. A comparison with functionally characterized Arabidopsis CLE protein sequences showed that many PtCLE proteins are closely related to their predicted Arabidopsis counterparts. Particularly, PtCLE3, PtCLE12, PtCLE14 and PtCLE38 comprised an identical CLE motif to AtCLE41/TDIF, which is known as a regulator of vascular cambium homeostasis, strongly supporting the idea that similar signaling pathways exist in both species to regulate wood formation and secondary growth. Transcriptome profiling revealed that PtCLE genes generally were differentially expressed while some PtCLE genes exhibited tissue-specific expression patterns. Moreover, compared to their Arabidopsis counterparts, PtCLE genes showed either similar or distinct expression patterns, implying functional conservation in some cases and functional divergence in others. CONCLUSIONS: Our study provides a genome-wide analysis of the CLE gene family in poplar, and highlights the potential roles of key PtCLE genes in the regulation of secondary growth and wood formation. The comparative analysis revealed that functional conservation may exist between PtCLEs and their AtCLE orthologues, which was further supported by transcriptomic analysis. Transcriptional profiling provided further insights into possible functional divergence, evidenced by differential expression patterns of various PtCLE genes.

CLV3-CLV1 signaling governs flower primordia outgrowth across environmental temperatures.

The initiation and outgrowth of floral primordia are critical for flower formation and reproductive success; however, the underlying mechanisms are still unclear. Two reports (Jones et al.; John et al.) shed light on how CLV3-CLV1 signaling promoted flower primordia formation and outgrowth by regulating auxin biosynthesis under distinct environmental temperatures.

CLE peptides in vascular development.

The plant vascular system consists of two conductive tissues, phloem and xylem. The vascular meristem, namely the (pro-)cambium, is a stem-cell tissue that gives rise to both xylem and phloem. Recent studies have revealed that CLAVATA3/Embryo Surrounding Region-related (CLE) peptides function in establishing the vascular system through interaction with phytohormones. In particular, TDIF/CLE41/CLE44, phloem-derived CLE peptides, promote the proliferation of vascular cambium cells and prevent them from differentiating into xylem by regulating WOX4 expression through the TDR/PXY receptor. In this review article, we outline recent advances on how CLE peptides function in vascular development in concert with phytohormones through mediating cell-cell communication. The perspective of CLE peptide signaling in vascular development is also discussed.

pH regulates peptide-receptor perception.

Diverse plant small peptides are perceived by their corresponding receptors to mediate local or long-distance intercellular communications in various developmental and adaptive programs; notably, the mechanisms of peptide-receptor perception remain largely unrevealed. Two reports (Liu et al.; Diaz-Ardila et al.) shed light on how pH regulates peptide-receptor perception.

CLE peptides join the plant longevity club.

Leaf senescence, the final step of leaf development, is an essential adaptive process that involves intricate regulatory networks mediated by various developmental and environmental clues. Two recent reports, by Zhang Z. et al. and Zhang Y. et al., shed light on how CLE peptides recruit reactive oxygen species (ROS) and ethylene signaling to promote plant leaf longevity.

Functional characterization of C-TERMINALLY ENCODED PEPTIDE (CEP) family in Brassica rapa L.

The small regulatory C-TERMINALLY ENCODED PEPTIDE (CEP) peptide family plays crucial roles in plant growth and stress response. However, little is known about this peptide family in Brassica species. Here, we performed a systematic analysis to identify the putative Brassica rapa L. CEP (BrCEP) gene family. In total, 27 BrCEP genes were identified and they were classified into four subgroups based on the CEP motifs similarity. BrCEP genes displayed distinct expression patterns in response to both developmental and several environmental signals, suggesting their broad roles during Brassica rapa development. Furthuremore, the synthetic BrCEP3 peptide accelerated Brassica rapa primary root growth in a hydrogen peroxide (H2O2) and Ca2+ dependent manner. In summary, our work will provide fundamental insights into the physiological function of CEP peptides during Brassica rapa development.

Systematic Annotation Reveals CEP Function in Tomato Root Development and Abiotic Stress Response.

Tomato (Solanum lycopersicum) is one of the most important vegetable crops worldwide; however, environmental stressors severely restrict tomato growth and yield. Therefore, it is of great interest to discover novel regulators to improve tomato growth and environmental stress adaptions. Here, we applied a comprehensive bioinformatics approach to identify putative tomato C-TERMINALLY ENCODED PEPTIDE (CEP) genes and to explore their potential physiological function in tomato root development and abiotic stress responses. A total of 17 tomato CEP genes were identified and grouped into two subgroups based on the similarity of CEP motifs. The public RNA-Seq data revealed that tomato CEP genes displayed a diverse expression pattern in tomato tissues. Additionally, CEP genes expression was differentially regulated by nitrate or ammonium status in roots and shoots, respectively. The differences in expression levels of CEP genes induced by nitrogen indicate a potential involvement of CEPs in tomato nitrogen acquisition. The synthetic CEP peptides promoted tomato primary root growth, which requires nitric oxide (NO) and calcium signaling. Furthermore, we also revealed that CEP peptides improved tomato root resistance to salinity. Overall, our work will contribute to provide novel genetic breeding strategies for tomato cultivation under adverse environments.

Genome-wide identification reveals the function of CEP peptide in cucumber root development.

C-Terminally Encoded (CEP) peptides are crucial plant growth regulators. Nevertheless, their physiological roles in cucumber (Cucumis sativus L.), an essential worldwide economical vegetable, remains untapped. In this study, 6 cucumber CEP (CsCEP) genes were identified. A comprehensive analysis showed that the CsCEP proteins displayed conserved characteristics to the identified CEP protein members in other species. CsCEP genes expression levels were variant in cucumber tissues, and were also differentially induced by several environmental factors, suggesting distinct and overlapping roles of CsCEPs in various cucumber developmental processes. We further revealed that synthetic CsCEP4 peptide promoted cucumber primary root growth in a reactive oxygen species (ROS) dependent manner. Overall, our work will provide fundamental insights into the crucial physiological roles of small bioactive peptides during cucumber root development.

Developmental roles of Auxin Binding Protein 1 in Arabidopsis thaliana.

Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.

Clathrin-mediated trafficking and PIN trafficking are required for auxin canalization and vascular tissue formation in Arabidopsis.

The flexible development of plants is characterized by a high capacity for post-embryonic organ formation and tissue regeneration, processes, which require tightly regulated intercellular communication and coordinated tissue (re-)polarization. The phytohormone auxin, the main driver for these processes, is able to establish polarized auxin transport channels, which are characterized by the expression and polar, subcellular localization of the PIN1 auxin transport proteins. These channels are demarcating the position of future vascular strands necessary for organ formation and tissue regeneration. Major progress has been made in the last years to understand how PINs can change their polarity in different contexts and thus guide auxin flow through the plant. However, it still remains elusive how auxin mediates the establishment of auxin conducting channels and the formation of vascular tissue and which cellular processes are involved. By the means of sophisticated regeneration experiments combined with local auxin applications in Arabidopsis thaliana inflorescence stems we show that (i) PIN subcellular dynamics, (ii) PIN internalization by clathrin-mediated trafficking and (iii) an intact actin cytoskeleton required for post-endocytic trafficking are indispensable for auxin channel formation, de novo vascular formation and vascular regeneration after wounding. These observations provide novel insights into cellular mechanism of coordinated tissue polarization during auxin canalization.