Spatio-temporal plant hormonomics: From tissue to subcellular resolution.

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.

Application of Long-Chained Auxin Conjugates Influenced Auxin Metabolism and Transcriptome Response in Brassica rapa L. ssp. pekinensis.

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.

Rapid profiling of cytokinins using supercritical fluid chromatography coupled with tandem mass spectrometry.

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.

Karrikinolide1 (KAR1), a Bioactive Compound from Smoke, Improves the Germination of Morphologically Dormant Apium graveolens L. Seeds by Reducing Indole-3-Acetic Acid (IAA) Levels.

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.

Chloroplast Auxin Efflux Mediated by ABCB28 and ABCB29 Fine-Tunes Salt and Drought Stress Responses in Arabidopsis.

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.