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Smoke-water (SW) and Karrikinolide1 (KAR1) release dormancy and improve seed germination in many plant species. Therefore, we tested SW (1:2500 v/v) and KAR1 (10-7 M) to break the morphological dormancy of celery cultivar (Apium graveolens L.). In the first trial, seeds were subjected to a 21-day incubation period at 20 °C with SW and KAR1 applied as single treatments. KAR1 showed significantly improved germination (30.7%) as compared to SW (17.2%) and a water control (14.7%). In seed soaking experiments, SW, KAR1, and gibberellic acid (GA3) treatments showed higher germination percentages than the water control after 3 and 6 h of soaking. However, prolonged soaking (12 h) reduced germination percentages for all treatments, indicating a detrimental effect. Analysis of KAR1 content dynamics in 7-day- and 21-day-old celery seeds indicated its prolonged effects on germination and dormancy alleviation. Phytohormones, including auxins in 7-day-old and cytokinins in 7-day- and 21-day-old celery seedlings, along with their precursors and metabolites, were analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) after treatment with KAR1 and SW. The analysis of auxin levels in 7-day-old seeds revealed a negative correlation between seed germination and auxin (indole-3-acetic acid, IAA) content. Notably, it was found that KAR1-treated seeds significantly reduced IAA levels in all treatments. SW and KAR1 did not significantly affect cytokinin levels during celery germination except for N6-Isopentenyladenine. Hence, further research is needed to understand their precise role in celery seed germination. This work will improve our understanding of the role of bioactive compounds from plant-derived smoke and how they regulate hormonal responses and improve germination efficiency in celery.
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Due to technological advances in mass spectrometry, significant progress has been achieved recently in plant hormone research. Nowadays, plant hormonomics is well established as a fully integrated scientific field focused on the analysis of phytohormones, mainly on their isolation, identification and spatiotemporal quantification in plants. This review represents a comprehensive meta-study of the advances in the phytohormone analysis by mass spectrometry over the past decade. To address current trends and future perspectives, Web of Science data were systematically collected and key features such as mass spectrometry-based analyses were evaluated using multivariate data analysis methods. Our findings showed that plant hormonomics is currently divided into targeted and untargeted approaches. Both aim to miniaturize the sample, allowing high-resolution quantification to be covered in plant organs as well as subcellular compartments. Therefore, we can study plant hormone biosynthesis, metabolism and signalling at a spatio-temporal resolution. Moreover, this trend has recently been accelerated by technological advances such as fluorescence-activated cell sorting or mass spectrometry imaging.
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BACKGROUND: The determination of plant hormones is still a very challenging analytical discipline, mainly due to their low concentration in complex plant matrices. Therefore, the involvement of very sensitive high-throughput techniques is required. Cytokinins (CKs) are semi-polar basic plant hormones regulating plant growth and development. Modern methods for CK determination are currently based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which enables the separation of CK isomeric forms occurring endogenously in plants. Here, ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC-MS/MS) was used for the simultaneous determination of 37 CK metabolites. RESULTS: The chromatographic conditions were tested on three different columns with various retention mechanisms. Hybrid silica modified with 2-picolylamine was selected as the stationary phase. Several parameters such as column temperature, back pressure regulation, mobile phase composition and make-up solvent were investigated to achieve efficient separation of CK isomers and reasonable sensitivity. Compared to UHPLC-MS/MS, a 9-min chromatographic analysis using a mobile phase of supercritical CO2 and 5 mM ammonia in methanol represents a three-fold acceleration of total run time. The quantification limit of UHPSFC-MS/MS method was in the range of 0.03-0.19 fmol per injection and the method validation showed high accuracy and precision (below 15 % for most analytes). The method was finally applied to the complex plant matrix of the model plant Arabidopsis thaliana and the obtained profiles of CK metabolites were compared with the results from the conventional UHPLC-MS/MS method. SIGNIFICANCE: The presented work offers a novel approach for quantification of endogenous CKs in plants. Compared to the conventional UHPLC-MS/MS, the total run time is shorter and the matrix effect lower for the key CK metabolites. This approach opens the opportunity to utilize UHPSFC-MS/MS instrumentation for targeted plant hormonomics including other plant hormone families.
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Cromatografía con Fluido Supercrítico , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Citocininas , Reguladores del Crecimiento de las Plantas , Cromatografía con Fluido Supercrítico/métodos , Cromatografía Líquida de Alta Presión/métodos , PlantasRESUMEN
Due to their long lifespan, trees and bushes develop higher order of branches in a perennial manner. In contrast to a tall tree, with a clearly defined main stem and branching order, a bush is shorter and has a less apparent main stem and branching pattern. To address the developmental basis of these two forms, we studied several naturally occurring architectural variants in silver birch (Betula pendula). Using a candidate gene approach, we identified a bushy kanttarelli variant with a loss-of-function mutation in the BpMAX1 gene required for strigolactone (SL) biosynthesis. While kanttarelli is shorter than the wild type (WT), it has the same number of primary branches, whereas the number of secondary branches is increased, contributing to its bush-like phenotype. To confirm that the identified mutation was responsible for the phenotype, we phenocopied kanttarelli in transgenic BpMAX1::RNAi birch lines. SL profiling confirmed that both kanttarelli and the transgenic lines produced very limited amounts of SL. Interestingly, the auxin (IAA) distribution along the main stem differed between WT and BpMAX1::RNAi. In the WT, the auxin concentration formed a gradient, being higher in the uppermost internodes and decreasing toward the basal part of the stem, whereas in the transgenic line, this gradient was not observed. Through modeling, we showed that the different IAA distribution patterns may result from the difference in the number of higher-order branches and plant height. Future studies will determine whether the IAA gradient itself regulates aspects of plant architecture.
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Ácidos Indolacéticos , Reguladores del Crecimiento de las Plantas , Árboles , Lactonas , Regulación de la Expresión Génica de las PlantasRESUMEN
Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum.
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Arabidopsis , Reguladores del Crecimiento de las Plantas , Reguladores del Crecimiento de las Plantas/metabolismo , Fluorescencia , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Hormonas/metabolismo , Raíces de Plantas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
N6-methyl adenosine (m6A) is a widespread internal mRNA modification impacting the expression of numerous genes. Here, we characterize auxin-related defects among the pleiotropic phenotypes of hypomorphic Arabidopsis thaliana mutants with impaired m6A status and reveal that they show strong resistance to exogenously applied auxin. By combining major published m6A datasets, we propose that among high-confidence target transcripts emerge those encoding the main components required for auxin signaling, including the TIR1/AFB auxin receptors and ARF transcriptional regulators. We also observe subtle changes in endogenous levels of indole-3-acetic acid metabolites in these hypomorphic lines, which correlate with the methylation status of indole-3-acetic acid amidohydrolase transcripts. In addition, we reveal that reduced m6A levels lead to defects in endodermal patterning in the primary root arising from impaired timing of periclinal cell divisions. These defects can be reverted by inhibition of auxin signaling. Together, our data underline that m6A likely affects auxin-dependent processes at multiple levels.
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Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.
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Auxin amino acid conjugates are considered to be storage forms of auxins. Previous research has shown that indole-3-acetyl-L-alanine (IAA-Ala), indole-3-propionyl-L-alanine (IPA-Ala) and indole-3-butyryl-L-alanine (IBA-Ala) affect the root growth of Brassica rapa seedlings. To elucidate the potential mechanism of action of the conjugates, we treated B. rapa seedlings with 0.01 mM IAA-, IPA- and IBA-Ala and investigated their effects on the auxin metabolome and transcriptome. IBA-Ala and IPA-Ala caused a significant inhibition of root growth and a decrease in free IAA compared to the control and IAA-Ala treatments. The identification of free auxins IBA and IPA after feeding experiments with IBA-Ala and IPA-Ala, respectively, confirms their hydrolysis in vivo and indicates active auxins responsible for a stronger inhibition of root growth. IBA-Ala caused the induction of most DEGs (807) compared to IPA-Ala (417) and IAA-Ala (371). All treatments caused similar trends in transcription profile changes when compared to control treatments. The majority of auxin-related DEGs were found after IBA-Ala treatment, followed by IPA-Ala and IAA-Ala, which is consistent with the apparent root morphology. In addition to most YUC genes, which showed a tendency to be downregulated, transcripts of auxin-related DEGs that were identified (UGT74E2, GH3.2, SAUR, IAA2, etc.) were more highly expressed after all treatments. Our results are consistent with the hypothesis that the hydrolysis of conjugates and the release of free auxins are responsible for the effects of conjugate treatments. In conclusion, free auxins released by the hydrolysis of all auxin conjugates applied affect gene regulation, auxin homeostasis and ultimately root growth inhibition.
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Brassica rapa , Gastrópodos , Animales , Ácidos Indolacéticos/farmacología , Brassica rapa/genética , Transcriptoma , Indoles , Alanina , Plantones/genéticaRESUMEN
Photosynthesis is among the first processes negatively affected by environmental cues and its performance directly determines plant cell fitness and ultimately crop yield. Primarily sites of photosynthesis, chloroplasts are unique sites also for the biosynthesis of precursors of the growth regulator auxin and for sensing environmental stress, but their role in intracellular auxin homeostasis, vital for plant growth and survival in changing environments, remains poorly understood. Here, we identified two ATP-binding cassette (ABC) subfamily B transporters, ABCB28 and ABCB29, which export auxin across the chloroplast envelope to the cytosol in a concerted action in vivo. Moreover, we provide evidence for an auxin biosynthesis pathway in Arabidopsis thaliana chloroplasts. The overexpression of ABCB28 and ABCB29 influenced stomatal regulation and resulted in significantly improved water use efficiency and survival rates during salt and drought stresses. Our results suggest that chloroplast auxin production and transport contribute to stomata regulation for conserving water upon salt stress. ABCB28 and ABCB29 integrate photosynthesis and auxin signals and as such hold great potential to improve the adaptation potential of crops to environmental cues.
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BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.
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Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.
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Apple species are the unique naturally rich source of dihydrochalcones, phenolic compounds with an elusive role in planta, but suggested auto-allelochemical features related to "apple replant disease" (ARD). Our aim was to elucidate the physiological basis of the phytotoxic action of dihydrochalcone phloretin in the model plant Arabidopsis and to promote phloretin as a new prospective eco-friendly phytotoxic compound. Phloretin treatment induced a significant dose-dependent growth retardation and severe morphological abnormalities and agravitropic behavior in Arabidopsis seedlings. Histological examination revealed a reduced starch content in the columella cells and a serious disturbance in root architecture, which resulted in the reduction in length of meristematic and elongation zones. Significantly disturbed auxin metabolome profile in roots with a particularly increased content of IAA accumulated in the lateral parts of the root apex, accompanied by changes in the expression of auxin biosynthetic and transport genes, especially PIN1, PIN3, PIN7, and ABCB1, indicates the role of auxin in physiological basis of phloretin-induced growth retardation. The results reveal a disturbance of auxin homeostasis as the main mechanism of phytotoxic action of phloretin. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD.
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Plant cell growth responds rapidly to various stimuli, adapting architecture to environmental changes. Two major endogenous signals regulating growth are the phytohormone auxin and the secreted peptides rapid alkalinization factors (RALFs). Both trigger very rapid cellular responses and also exert long-term effects [Du et al., Annu. Rev. Plant Biol. 71, 379-402 (2020); Blackburn et al., Plant Physiol. 182, 1657-1666 (2020)]. However, the way, in which these distinct signaling pathways converge to regulate growth, remains unknown. Here, using vertical confocal microscopy combined with a microfluidic chip, we addressed the mechanism of RALF action on growth. We observed correlation between RALF1-induced rapid Arabidopsis thaliana root growth inhibition and apoplast alkalinization during the initial phase of the response, and revealed that RALF1 reversibly inhibits primary root growth through apoplast alkalinization faster than within 1 min. This rapid apoplast alkalinization was the result of RALF1-induced net H+ influx and was mediated by the receptor FERONIA (FER). Furthermore, we investigated the cross-talk between RALF1 and the auxin signaling pathways during root growth regulation. The results showed that RALF-FER signaling triggered auxin signaling with a delay of approximately 1 h by up-regulating auxin biosynthesis, thus contributing to sustained RALF1-induced growth inhibition. This biphasic RALF1 action on growth allows plants to respond rapidly to environmental stimuli and also reprogram growth and development in the long term.
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Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Hormonas Peptídicas , Raíces de Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Hormonas Peptídicas/metabolismo , Fosfotransferasas , Raíces de Plantas/crecimiento & desarrolloRESUMEN
The factors underlying the plant collapse of certain melon-pumpkin graft combinations are not fully understood. Our working hypothesis was that impairment of photoassimilates transport in incompatible combinations induces an imbalance in the homeostasis of root auxin (indole-3-acetic acid; IAA) and of cytokinins, probably triggering plant collapse. Root IAA and cytokinins levels in the presence and absence of fruit and changes in root and scion metabolites were investigated in compatible and incompatible combinations. We showed elevated levels of IAA, 2-oxoindole-3-acetic acid (IAA catabolite), indole-3-acetylaspartate (IAA conjugate), and cis-zeatin-type cytokinins, but low levels of trans-zeatin-type cytokinins in the roots of plants of the incompatible combination during fruit ripening. Similarly, during fruit ripening, the expression of the YUCCA genes, YUC2, YUC6, and YUC11 (required for auxin biosynthesis), the GRETCHEN-HAGEN3 gene (required for auxin conjugation), and the cytokinin oxidase/dehydrogenase 7 (CKX7) gene (regulates the irreversible degradation of cytokinin) was enhanced in the roots of plants of the incompatible combination. Moreover, in the incompatible combination the fruiting process restricted transport of photoassimilates to the rootstock and induces their accumulation in the scion. In addition, high levels of hydrogen peroxide and malondialdehyde and reduced activity of antioxidant enzymes were observed in the roots of the incompatible graft. Our results showed that the collapse of the incompatible graft combination during fruit ripening is closely associated with a dramatic accumulation of IAA in the roots, which probably elicits oxidative damage and disturbs the balance of IAA and cytokinins that is of critical importance in melon-pumpkin graft compatibility.
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Brassica napus (rapeseed) is the second most important oilseed crop worldwide. Global rise in average ambient temperature and extreme weather severely impact rapeseed seed yield. However, fewer research explained the phenotype changes caused by moderate-to-high temperatures in rapeseed. To investigate these events, we determined the long-term response of three spring cultivars to different temperature regimes (21/18°C, 28/18°C, and 34/18°C) mimicking natural temperature variations. The analysis focused on the plant appearance, seed yield, quality and viability, and embryo development. Our microscopic observations suggest that embryonic development is accelerated and defective in high temperatures. Reduced viable seed yield at warm ambient temperature is due to a reduced fertilization rate, increased abortion rate, defective embryonic development, and pre-harvest sprouting. Reduced auxin levels in young seeds and low ABA and auxin levels in mature seeds may cause embryo pattern defects and reduced seed dormancy, respectively. Glucosinolates and oil composition measurements suggest reduced seed quality. These identified cues help understand seed thermomorphogenesis and pave the way to developing thermoresilient rapeseed.
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Fluctuating environmental conditions trigger adaptive responses in plants, which are regulated by phytohormones. During photoperiod stress caused by a prolongation of the light period, cytokinin (CK) has a protective function. Auxin often acts as an antagonist of CK in developmental processes and stress responses. Here, we investigated the regulation of the photoperiod stress response in Arabidopsis thaliana by auxin and its interaction with CK. Transcriptome analysis revealed an altered transcript abundance of numerous auxin metabolism and signaling genes after photoperiod stress treatment. The changes appeared earlier and were stronger in the photoperiod-stress-sensitive CK receptor mutant arabidopsis histidine kinase 2 (ahk2),3 compared to wild-type plants. The concentrations of indole-3-acetic acid (IAA), IAA-Glc and IAA-Asp increased in both genotypes, but the increases were more pronounced in ahk2,3. Genetic analysis revealed that the gain-of-function YUCCA 1 (YUC1) mutant, yuc1D, displayed an increased photoperiod stress sensitivity. In contrast, a loss of the auxin receptors TRANSPORT-INHIBITOR-RESISTANT 1 (TIR1), AUXIN SIGNALING F-BOX 2 (AFB2) and AFB3 in wild-type and ahk2,3 background caused a reduced photoperiod stress response. Overall, this study revealed that auxin promotes response to photoperiod stress antagonizing the protective CK.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citocininas/metabolismo , Citocininas/farmacología , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacología , Fotoperiodo , Raíces de Plantas/metabolismoRESUMEN
Indole-3-acetic acid (IAA) controls a plethora of developmental processes. Thus, regulation of its concentration is of great relevance for plant performance. Cellular IAA concentration depends on its transport, biosynthesis and the various pathways for IAA inactivation, including oxidation and conjugation. Group II members of the GRETCHEN HAGEN 3 (GH3) gene family code for acyl acid amido synthetases catalysing the conjugation of IAA to amino acids. However, the high degree of functional redundancy among them has hampered thorough analysis of their roles in plant development. In this work, we generated an Arabidopsis gh3.1,2,3,4,5,6,9,17 (gh3oct) mutant to knock out the group II GH3 pathway. The gh3oct plants had an elaborated root architecture, showed an increased tolerance to different osmotic stresses, including an IAA-dependent tolerance to salinity, and were more tolerant to water deficit. Indole-3-acetic acid metabolite quantification in gh3oct plants suggested the existence of additional GH3-like enzymes in IAA metabolism. Moreover, our data suggested that 2-oxindole-3-acetic acid production depends, at least in part, on the GH3 pathway. Targeted stress-hormone analysis further suggested involvement of abscisic acid in the differential response to salinity of gh3oct plants. Taken together, our data provide new insights into the roles of group II GH3s in IAA metabolism and hormone-regulated plant development.
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Arabidopsis , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Hormonas/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Salinidad , Agua/metabolismoRESUMEN
Soil salinization is increasing globally, driving a reduction in crop yields that threatens food security. Salinity stress reduces plant growth by exerting two stresses on plants: rapid shoot ion-independent effects which are largely osmotic and delayed ionic effects that are specific to salinity stress. In this study we set out to delineate the osmotic from the ionic effects of salinity stress. Arabidopsis thaliana plants were germinated and grown for two weeks in media supplemented with 50, 75, 100, or 125 mM NaCl (that imposes both an ionic and osmotic stress) or iso-osmolar concentrations (100, 150, 200, or 250 mM) of sorbitol, that imposes only an osmotic stress. A subsequent transcriptional analysis was performed to identify sets of genes that are differentially expressed in plants grown in (1) NaCl or (2) sorbitol compared to controls. A comparison of the gene sets identified genes that are differentially expressed under both challenge conditions (osmotic genes) and genes that are only differentially expressed in plants grown on NaCl (ionic genes, hereafter referred to as salt-specific genes). A pathway analysis of the osmotic and salt-specific gene lists revealed that distinct biological processes are modulated during growth under the two conditions. The list of salt-specific genes was enriched in the gene ontology (GO) term "response to auxin." Quantification of the predominant auxin, indole-3-acetic acid (IAA) and IAA biosynthetic intermediates revealed that IAA levels are elevated in a salt-specific manner through increased IAA biosynthesis. Furthermore, the expression of NITRILASE 2 (NIT2), which hydrolyses indole-3-acetonitile (IAN) into IAA, increased in a salt-specific manner. Overexpression of NIT2 resulted in increased IAA levels, improved Na:K ratios and enhanced survival and growth of Arabidopsis under saline conditions. Overall, our data suggest that auxin is involved in maintaining growth during the ionic stress imposed by saline conditions.
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The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, where it acts via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad-scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL-like (WOX) proteins in bryophytes. The relationship between CLAVATA, hormone homeostasis and the evolution of land plant stem cell functions is unknown. Here we show that in the moss, Physcomitrella (Physcomitrium patens), CLAVATA affects stem cell activity by modulating hormone homeostasis. CLAVATA pathway genes are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching, plant spread and auxin synthesis. The receptor-like kinase PpRPK2 plays the major role, and Pprpk2 mutants have abnormal responses to cytokinin, auxin and auxin transport inhibition, and show reduced expression of PIN auxin transporters. We propose a model whereby PpRPK2 modulates auxin gradients in filaments to determine stem cell identity and overall plant form. Our data indicate that CLAVATA-mediated auxin homeostasis is a fundamental property of plant stem cell function, probably exhibited by the last shared common ancestor of land plants.