Ready for regrowth : A physiological and metabolic characterization of young sugar beets under temporary drought

Although the sugar beet belongs to the rather drought relevant species, water availability plays a crucial role in terms of plant development and yield formation, hence water deficits can lead to adverse consequences. Aim of this work was the physiological and metabolic characterization of young temporarily drought-stressed sugar beets with special emphasis of the recovery process of shoots and roots under rewatering and possible differences thereby. In this work the analysis of the chronological order of physiological and metabolic alterations under drought and rewatering was studied and further how these changes were related to a phenotypic approach, namely infrared thermography (IRT). Besides this, an untargeted 1H nuclear magnetic resonance spectroscopy (1H-NMR) and targeted enzyme based metabolite assays were used for the identification and characterization of major metabolites of the primary metabolism aiming at the identification of the metabolic strategy of temporarily drought-stressed sugar beets. While the experimental setup allowed reproducible greenhouse experiments, the analytic approach has been optimized for both, small scale and high throughput analysis. Within the phenotypic approach using IRT the initial impairment of transpiration as first reaction to drought. However, stress-induced metabolic adaptations with subsequent membrane-destabilization and cellular damage were only detectable by the combined application of invasive and non-invasive methods. Only the combination of both techniques allowed the holistic analysis of drought-induced alterations with close attention to plant water status and osmotic adjustment. The untargeted 1H-NMR-analysis revealed clear stress-induced changes of the primary metabolism and its reprogramming under rewatering. While drought lead rather to a downregulation of glycolysis and TCA-cycle in shoots and roots, amino acids generally increased. The observed distinct dynamics of shoots and roots under rewatering might be ascribed to the different functions of both organs. It can be concluded that the reactions to drought and rewatering are distinct and organ-specific processes that are actively driven by the plant. Moreover, the recovery process does not seem to be only the de-acclimation of the stress and is thus not the simple return to initial control conditions. The presented results contribute to a better understanding of the physiological and metabolic alterations under temporary drought-stress and rewatering in sugar beet and they provide valuable information for the breeding of drought-tolerant species while the applied analytical methods enables a quick and reliable high-throughput metabolite analysis.Obwohl die Zuckerrübe zu den eher trockentoleranten Spezies zählt, spielt Wasser bezüglich Entwicklung und Ertragsbildung dennoch eine entscheidende Rolle und eine Limitierung kann zu erheblichen negativen Folgen führen. Ziel dieser Arbeit war die physiologische und metabolische Charakterisierung von jungen Zuckerrüben unter temporärem Trockenstress mit besonderem Fokus auf den Recovery-Prozess von Spross und Wurzel unter Wiederbewässerung und mögliche Unterschiede dabei. Es erfolgte eine Analyse der chronologischen Abläufe von physiologischen und metabolischen Veränderungen unter Trockenstress und Wiederbewässerung, und wie diese im Zusammenhang mit einem phänotypischen Ansatz, der Infrarot-thermographie (IRT), stehen. Darüber hinaus wurde eine Identifizierung und Charakterisierung von Hauptmetaboliten des Primärstoffwechsels zur Untersuchung der metabolischen Strategie von temporär gestressten Zuckerüben mittels Kernspinresonanzspektroskopie (1H nuclear magnetic resonance spectroscopy, 1H-NMR) und enzymbasierten Metabolitanalysen durchgeführt. Der experimentelle Ansatz ermöglichte robuste und reproduzierbare Versuche unter Gewächshausbedingen und die Analytik wurde so optimiert, dass sie sowohl für den Einsatz von kleineren Probenmengen als auch für die Hochdurchsatzanalytik geeignet war. In dem phänotypischen Ansatz, konnte mittels IRT die initiale Beeinträchtigung der Transpiration als erste Reaktion auf Trockenstress festgehalten werden. Die metabolischen Anpassungen auf den Stress mit anschließender Membran-Destabilisierung und Zellschädigung konnten jedoch nur durch die Kombination aus invasiven und nicht-invasiven Verfahren aufgedeckt werden. Nur die Kombination beider Techniken ermöglichte eine ganzheitliche Beurteilung trockenstress-induzierter Veränderungen mit Fokus auf den Wasserhaushalt und die osmotische Anpassung. Die 1H-NMR Analyse legte eindeutige stress-induzierte Veränderungen des Primärstoffwechsels und dessen Umprogrammierung unter Wiederbewässerung offen. Während Trockenheit eher zu einer Herabregulation von Glykolyse und Citratzyklus in Spross und Wurzel führte, reagierten die Aminosäuren mit einem generellen Anstieg. Unter Wiederbewässerung zeigten beide Organe jedoch eine unterschiedliche Dynamik in der Erholungsreaktion, die vermutlich auf die unterschiedlichen Funktionen von Spross und Wurzel zurückzuführen sind. Es handelt sich bei den Reaktionen auf Trockenheit und Wiederbewässerung offenbar um unterschiedliche und organ-spezifische Prozesse. Darüber hinaus scheint der Erholungsprozess keine simple De-Akklimatisierung des Stresses zu sein und ist damit nicht nur die Rückkehr zu Kontrollbedingungen. Die Ergebnisse dieser Arbeit tragen zu einem verbesserten Verständnis der physiologischen und metabolischen Veränderungen unter temporärem Trockenstress und Wiederbewässerung bei und liefern wertvolle Informationen für die Züchtung trockentoleranter Sorten. Die angewendeten Methoden bieten die Möglichkeit auch im Hochdurchsatzverfahren eine schnelle und zuverlässige Analyse durchzuführen

A Protein-Linger Strategy Keeps the Plant On-Hold After Rehydration of Drought-Stressed Beta vulgaris

Most crop plants are exposed to intermittent drought periods. To cope with these continuous changes, plants need strategies to prevent themselves from exhaustive adjustment maneuvers. Drought stress recovery has been shown to be an active process, possibly involved in a drought memory effect allowing plants to better cope with recurrent aridity. An integrated understanding of the molecular processes of enhanced drought tolerance is required to tailor key networks for improved crop protection. During summer, prolonged periods of drought are the major reason for economic yield losses of sugar beet (Beta vulgaris) in Europe. A drought stress and recovery time course experiment was carried out under controlled environmental conditions. In order to find regulatory key mechanisms enabling plants to rapidly react to periodic stress events, beets were either subjected to 11 days of progressive drought, or were drought stressed for 9 days followed by gradual rewatering for 14 days. Based on physiological measurements of leaf water relations and changes in different stress indicators, plants experienced a switch from moderate to severe water stress between day 9 and 11 of drought. The leaf proteome was analyzed, revealing induced protein pre-adjustment (prior to severe stress) and putative stress endurance processes. Three key protein targets, regulatory relevant during drought stress and with lingering levels of abundance upon rewatering were further exploited through their transcript performance. These three targets consist of a jasmonate induced, a salt-stress enhanced and a phosphatidylethanolamine-binding protein. The data demonstrate delayed protein responses to stress compared to their transcripts and indicate that the lingering mechanism is post-transcriptionally regulated. A set of lingering proteins is discussed with respect to a possible involvement in drought stress acclimation and memory effects

1H-NMR metabolomic profiling reveals a distinct metabolic recovery response in shoots and roots of temporarily drought-stressed sugar beets.

Yield formation in regions with intermittent drought periods depends on the plant's ability to recover after cessation of the stress. The present work assessed differences in metabolic recovery of leaves and roots of drought-stressed sugar beets with high temporal resolution. Plants were subjected to drought for 13 days, and rewatered for 12 days. At one to two-day intervals, plant material was harvested for untargeted 1H-NMR metabolomic profiling, targeted analyses of hexose-phosphates, starch, amino acids, nitrate and proteins, and physiological measurements including relative water content, osmotic potential, electrolyte leakage and malondialdehyde concentrations. Drought triggered changes in primary metabolism, especially increases in amino acids in both organs, but leaves and roots responded with different dynamics to rewatering. After a transient normalization of most metabolites within 8 days, a second accumulation of amino acids in leaves might indicate a stress imprint beneficial in upcoming drought events. Repair mechanisms seemed important during initial recovery and occurred at the expense of growth for at least 12 days. These results indicate that organ specific metabolic recovery responses might be related to distinct functions and concomitant disparate stress levels in above- and belowground organs. With respect to metabolism, recovery was not simply a reversal of the stress responses

Plant water status and indicators of membrane damage of sugar beet leaves.

<p>Relative water content (RWC, A) osmotic potential (OP, B), electrolyte leakage (EL, C) and malondialdehyde (MDA) concentrations (D) of leaves under regular water supply (closed circles) and temporary drought (open circles). The area plot represents the gravimetric relative soil water content (SWC w/w %). Plants were rewatered after 13 d as indicated by the dashed line. All values are means ± s.e. (n = 4). Significant differences to the control plants (Duncan, α = 0.5) are indicated by *P < 0.05.</p

Chemical shifts used for identification and quantification of amino acids, other amino compounds and other compounds in ¹H-NMR spectra of beet root and leaf polar extracts (in D₂O, pH_apparent 6.0), expressed as relative values to the TSP resonance at 0 ppm.

<p>s: singlet, d: doublet, dd: doublet of doublets, t: triplet, m: multiplet.</p

Result of the principal component analysis (PCA) of the ¹H-NMR profiles.

<p>Scores plot of leaf (A) and root (C), loadings plot of leaf (B) and root (D); samples size n = 3. The PC1 x PC2 plots represent 53.6% and 68.3% of total variance for leaves and roots, respectively. In the scores plot circles represent control plants and triangles represents temporary stressed plants. Trajectories in the scores plot represent the temporal development of metabolic response during the treatments (blue arrow, control; bicolored arrow: red: drought, green: rewatered. Abbreviations: Asn, asparagine; Asp, asparagine; Gaba, γ-aminobutyric acid; GB, glycine betaine; Gln, glutamine; Glu, glutamate; Ile, isoleucine; Leu, leucine; Val, valine; Phe, phenylalanine; Pro, proline; Pyro-Glu, pyroglutamate; Ser, serine; Trp, tryptophan; Tyr, tyrosine; UDPG-like, uridine diphosphate glucose-like; unk, unknown compound.</p

Change of biomass of sugar beet shoots and roots.

<p>Plant dry weight of sugar beets shoots (A) and roots (B) of control (closed circles) and rewatered (open circles) plants. Plants were rewatered after 13 d as indicated by the dashed line. The area plot represents the gravimetric relative soil water content (SWC w/w %). All values are means ± s.e. (n = 4). Significant differences to the control plants (Duncan, α = 0.5) are indicated by *P < 0.05.</p