Molecular Plant Physiology
We use the model plant Arabidopsis thaliana to explore how plants organize growth and water relations, and how they handle attacks by pathogens.
We use the model plant Arabidopsis thaliana to explore how plants organize growth and water relations, and how they handle attacks by pathogens.
Our research focuses on
Aquaporins and plant water relations
Aquaporins or major intrinsic proteins (MIP) are universal membrane proteins, which facilitate the passage of water and other small, uncharged molecules. Plant genomes encode more than 30 MIP isoforms. We focus on the regulation plasma membrane intrinsic proteins (PIP) and their role in plant root development and in response to stress conditions.
Small-molecule glycosyltransferases (UGTs) in plant dense and signaling
Numerous endogenous molecules are involved in plant defense and signaling. Their biosynthesis and activity is frequently controlled by conjugation with carbohydrates. We are interested in UGTs and their substrates affecting plant pathogen defense, in particular related to the immune-stimulating salicylic acid, N-hydroxy pipecolic acid, and isoleucic acid.
Microtubular control of cell expansion
Plants have to coordinate cellular expansions to establish their final form. The mutation of TORTIFOLIA (TOR) genes leads to a consistent, either right- or left-handed torsional growth of leaf petioles. tor mutations affect a microtubule-associated protein (tor1) or amino acid exchanges in tubulin subunits (tor2, tor3). Mutated tubulins also affect the left-right asymmetry in animal body plans.
Functions and physiological roles of plant aquaporins
Water relations are fundamental to sustainable plant growth and productivity, which are frequently challenged by reduced water availability and to developmental processes. Aquaporins are integral membrane proteins of the major intrinsic protein (MIP) family, which permeabilize membranes for the passage of water driven by osmotic or hydrostatic pressure, although individual isoforms may allow the exchange of other small, uncharged molecules. In 1994, the laboratory of Maarten Chrispeels and we independently discovered the first plasma membrane aquaporins in plants; accordingly, we named them PIP, plasma membrane intrinsic protein(s) [Kammerloher et al. (1994) Plant J. 6, 187-199].
The model plant Arabidopsis thaliana as well as other plants encode more than 30 MIP isoforms, 10-15 of those belonging to the PIP subfamily. PIPs constitute the most highly conserved MIP subfamily, yet they still split into two clearly distinguishable subgroups PIP1 and PIP2 in all higher plants.
We focus on the analyses of the complement of 13 Arabidopsis PIPs and their role in plant water relations (root-shoot water relocation, water use efficiency), their impact and regulation in response to stress conditions (reduced water availability due to drought and heat), their regulation at the protein level, and their function in developmental processes (root development).
In collaboration with the groups of Christophe Maurel (SupAgro, INRA Montpellier, France) and Malcolm Bennett (CPIB, University of Nottingham, UK) we discovered that PIPs, in particular PIP2;1, are involved in controlling tissue hydraulics during the development and outgrowth of lateral root primordia which have to penetrate the overlaying cell layers. PIPs, although having different spatiotemporal expression patterns enhances the rate of lateral root emergence [Péret, Li, Zhao, Band et al. (2012) Nature Cell Biol. 14, 991-998].
Small-molecule glycosyltransferases in plant defense and signaling
Plants utilize a plethora of small organic molecules for signaling in development and defense. These metabolites are frequently conjugated by hydrophilic moieties to control their activity, transport, or localization. Among these reactions, glycosylation plays a prominent role. Thus, plants have evolved a diverse set of glycosylation reactions that is reflected by the presence >100 isoforms of UDP-carbohydrate-dependent glycosyltransferases (UGTs) in their genomes. However, most of them are orphan enzymes without known endogenous substrates and physiological functions.
A systems biology approach based on genetics, plant-pathogen interactions, metabolomics, and biochemistry revealed a central role of UGT76B1 as a metabolic hub regulating three immune-stimulatory compounds. UGT76B1 glucosylates and inactivates salicylic acid (SA), N-hydroxypipecolic acid (NHP), and isoleucic acid (ILA) in a mutually competitive manner. Plants deficient in UGT76B1 have a constitutively activated basal, systemic acquired resistance- (SAR)-like defense, which is dependent on the NHP formation, yet eventually executed by SA. UGT76B1 mutually keeps NHP, SA, and ILA in balance and thereby “keeps immune response in check“ (H. Hõrak, Plant Cell (In Brief) 33, 2021). UGT76B1 overexpression represses free NHP and abolishes SAR.
ILA was identified as a substrate of UGT76B1 by a non-targeted metabolome approach without any prior clue on its chemical nature.
In addition, we investigated the complex glycosylation patterns of flavonols and their role in flavonol biosynthesis and plant development. UGT78D1 and UGT78D2 are the major isoforms responsible for the initial conjugation at the 3-O-position. The lack of 3-O-glycosylation leads to a feedback inhibition of flavonol aglycon biosynthesis. Flavonols have been long studied for their implication in polar auxin transport in plant development; however the nature of bioactive compound(s) in planta has been elusive. Based on a genetic approach using mutants affecting flavonol biosynthesis and glycosylation, we identified kaempferol 3-O-rhamnoside-7-O-rhamnoside as an endogenous inhibitor of polar auxin transport in Arabidopsis shoots.
How to grow straight: microtubular involvement in the control of directed organ and single cell expansion
Gerhard Röbbelen at the University of Göttingen, Germany performed pioneering genetic studies of Arabidopsis thaliana back in the 1940s. His student Erna Reinholz named one of the identified mutants tortifolia (now tor1), because it displayed right-handed, torsional growth of the leaves due to a twisting of the leaf stalks (Reinholz, 1947, FIAT Report 1006, 33-34). Five decades later, this mutant served as the lead for the identification of independent torsional mutants, which were assigned to three complementation groups: tor1 and tor2 lead to consistently right-handed torsions, whereas tor 3 shows left-handed torsional growth.
TOR1 is a plant-specific microtubule-associated protein that is involved in a fundamental control of straight growth of plant organs: its loss leads to an altered, left-oriented net orientation of otherwise oblique cortical microtubules in elongating cells, which translates to right-handed expansion growth.
Structural changes in tubulin subunits themselves may alter the dynamicity of cortical microtubules, which also may affect the directional growth of single cells and organs. tor2 depends on the change of the conserved Arg2 of α-TUBULIN 4 into Lys. This may interrupt hydrogen bonds normally formed between α- and β-tubulin and potentially affects the GTPase region of the β-tubulin. Helical growth of tor2 arises independently from the cellular context, but also in isolated, single cells (trichomes, cell suspension cells) derived from the mutant. Thus, torsional growth of an organ may result from a higher order expression of the helical expansion of individual cells.
Several additional tubulin point mutations exerting helical growth patterns were identified by Japanese colleagues (Ishida et al., 2007, PNAS 104, 8544). Cytoskeletal and in particular tubulin mutants have been shown by other groups to affect the established left-right asymmetry across kingdoms [e.g. Lobikin et al. (2012) PNAS 109, 12586].
The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis strain SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic ABA-dependent manner. Application of SA190 priming on crops is demonstrated for alfalfa, showing enhanced performance under drought conditions. In summary, a single beneficial root bacterial strain can help plants to resist drought conditions.
Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used RNA-sequencing after ribosomal RNA depletion to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0. Concerning the changes detected in the organellar transcriptomes, chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Conversely, alternative splicing (AS) affected nearly 1,500 genes (9% of expressed nuclear genes). Of these, 42% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Moreover, we identified 927 isoform switching events. We provide a table of the most interesting candidates, and as proof of principle, increased drought tolerance of the carbonic anhydrase ca1 and ca2 mutants is shown. In addition, altering the relative contributions of the spliced isoforms could increase drought resistance. For example, our data suggest that the accumulation of a nonfunctional FLM (FLOWERING LOCUS M) isoform and not the ratio of FLM-ß and -δ isoforms may be responsible for the phenotype of early flowering under long-day drought conditions. In sum, our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought.
Defense responses in plants are based on complex biochemical processes. Systemic acquired resistance (SAR) helps to fight infections by (hemi-)biotrophic pathogens. One important signaling molecule in SAR is pipecolic acid (Pip), accumulation of which is dependent on the aminotransferase ALD1 in Arabidopsis. While exogenous Pip primes de- fense responses in the monocotyledonous cereal crop barley (Hordeum vulgare), it is currently unclear if endogenous Pip plays a role in disease resistance in monocots. Here, we generated barley ald1 mutants using CRISPR/Cas9, and assessed their capacity to mount SAR. Endogenous Pip levels were reduced after infection of the ald1 mutant, and this altered systemic defense against the fungus Blumeria graminis f. sp. hordei. Furthermore, Hvald1 plants did not emit nonanal, one of the key volatile compounds that are normally emitted by barley plants after the activation of SAR. This resulted in the inability of neighboring plants to perceive and/or respond to airborne cues and prepare for an up- coming infection, although HvALD1 was not required in the receiver plants to mediate the response. Our results high- light the crucial role of endogenous HvALD1 and Pip for SAR, and associate Pip, in particular together with nonanal, with plant-to-plant defense propagation in the monocot crop barley.
Glucosylation modulates the biological activity of small molecules and frequently leads to their inactivation. The Arabidopsis thaliana glucosyltransferase UGT76B1 is involved in conjugating the stress hormone salicylic acid (SA) as well as isoleucic acid (ILA). Here, we show that UGT76B1 also glucosylates N-hydroxypipecolic acid (NHP), which is synthesized by FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) and activates systemic acquired resistance (SAR). Upon pathogen attack, Arabidopsis leaves generate two distinct NHP hexose conjugates, NHP-O-b-glucoside and NHP glucose ester, whereupon only NHP-O-b-glucoside formation requires a functional SA pathway. The ugt76b1 mutants speciﬁcally fail to generate the NHP-O-b-glucoside, and recombinant UGT76B1 synthesizes NHP-O-b-glucoside in vitro in competition with SA and ILA. The loss of UGT76B1 elevates the endogenous levels of NHP, SA, and ILA and establishes a constitutive SAR-like immune status. Introgression of the fmo1 mutant lacking NHP biosynthesis into the ugt76b1 background abolishes this SAR-like resistance. Moreover, overexpression of UGT76B1 in Arabidopsis shifts the NHP and SA pools toward O-b-glucoside formation and abrogates pathogen-induced SAR. Our results further indicate that NHP-triggered immunity is SA-dependent and relies on UGT76B1 as a common metabolic hub. Thereby, UGT76B1-mediated glucosylation controls the levels of active NHP, SA, and ILA in concert to balance the plant immune status.
Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and antioxidant in eukaryotes. The recent discovery that kaempferol serves as a precursor for ubiquinone's benzenoid moiety both challenges the conventional view of flavonoids as specialized metabolites, and offers new prospects for engineering ubiquinone in plants. Here, we present evidence that Arabidopsis thaliana mutants lacking kaempferol 3-O-rhamnosyltransferase (ugt78d1) and kaempferol 3-O-glucosyltransferase (ugt78d2) activities display increased de novo biosynthesis of ubiquinone and increased ubiquinone content. These data are congruent with the proposed model that unprotected C-3 hydroxyl of kaempferol triggers the oxidative release of its B-ring as 4-hydroxybenzoate, which in turn is incorporated into ubiquinone. Ubiquinone content in the ugt78d1/ugt78d2 double knockout represented 160% of wild-type level, matching that achieved via exogenous feeding of 4-hydroxybenzoate to wild-type plants. This suggests that 4-hydroxybenzoate is no longer limiting ubiquinone biosynthesis in the ugt78d1/ugt78d2 plants. Evidence is also shown that the glucosylation of 4-hydroxybenzoate as well as the conversion of the immediate precursor of kaempferol, dihydrokaempferol, into dihydroquercetin do not compete with ubiquinone biosynthesis in A. thaliana.
Bamboos, member of the family Poaceae, represent many interesting features with respect to their fast and extended vegetative growth, unusual, yet divergent flowering time across species, and impact of sudden, large scale flowering on forest ecology. However, not many studies have been conducted at the molecular level to characterize important genes that regulate vegetative and flowering habit in bamboo. In this study, two bamboo FD genes, BtFD1 and BtFD2, which are members of the florigen activation complex (FAC) have been identified by sequence and phylogenetic analyses. Sequence comparisons identified one important amino acid, which was located in the DNA-binding basic region and was altered between BtFD1 and BtFD2 (Ala146 of BtFD1 vs. Leu100 of BtFD2). Electrophoretic mobility shift assay revealed that this alteration had resulted into ten times higher binding efficiency of BtFD1 than BtFD2 to its target ACGT motif present at the promoter of the APETALA1 gene. Expression analyses in different tissues and seasons indicated the involvement of BtFD1 in flower and vegetative development, while BtFD2 was very lowly expressed throughout all the tissues and conditions studied. Finally, a tenfold increase of the AtAP1 transcript level by p35S::BtFD1 Arabidopsis plants compared to wild type confirms a positively regulatory role of BtFD1 towards flowering. However, constitutive expression of BtFD1 had led to dwarfisms and apparent reduction in the length of flowering stalk and numbers of flowers/plant, whereas no visible phenotype was observed for BtFD2 overexpression. This signifies that timely expression of BtFD1 may be critical to perform its programmed developmental role in planta.
Plants are constantly exposed to stressful environmental conditions. Plant stress reactions were mainly investigated for single stress factors. However, under natural conditions plants may be simultaneously exposed to different stresses. Responses to combined stresses cannot be predicted from the reactions to the single stresses. Flavonoids accumulate in Arabidopsis thaliana during exposure to UV-A, UV-B or cold, but the interactions of these factors on ﬂavonoid biosyn- thesis were unknown. We therefore investigated the interaction of UV radiation and cold in regulating the expression of well-characterized stress-regulated genes, and on transcripts and metabolites of the ﬂavonoid biosynthetic path- way in 52 natural Arabidopsis accessions that differ widely in their freezing tolerance. The data revealed interactions of cold and UV on the regulation of stress-related and ﬂavonoid biosynthesis genes, and on ﬂavonoid composition. In many cases, plant reactions to a combination of cold and UV were unique under combined stress and not predictable from the responses to the single stresses. Strikingly, all correlations between expression levels of ﬂavonoid biosynthesis genes and ﬂavonol levels were abolished by UV-B exposure. Similarly, correlations between transcript levels of ﬂavonoid biosynthesis genes or ﬂavonoid contents, and freezing tolerance were lost in the presence of UV radiation, while correlations with the expression levels of cold-regulated genes largely persisted. This may indicate different molecular cold acclimation responses in the presence or absence of UV radiation.
Throughout the temperate zones, plants face combined drought and heat spells in increasing frequency and intensity. Here, we compared periodic (intermittent, i.e., high-frequency) versus chronic (continuous, i.e., high-intensity) drought-heat stress scenarios in gray poplar (Populus3 canescens) plants for phenotypic and transcriptomic effects during stress and after recovery. Photosynthetic productivity after stress recovery exceeded the performance of poplar trees without stress experience. We analyzed the molecular basis of this stress-related memory phenotype and investigated gene expression responses across ﬁve major tree compartments including organs and wood tissues. For each of these tissue samples, transcriptomic changes induced by the two stress scenarios were highly similar during the stress phase but strikingly divergent after recovery. Characteristic molecular response patterns were found across tissues but involved different genes in each tissue. Only a small fraction of genes showed similar stress and recovery expression proﬁles across all tissues, including type 2C protein phosphatases, the LATE EMBRYOGENESIS ABUNDANT PROTEIN4-5 genes, and homologs of the Arabidopsis (Arabidopsis thaliana) transcription factor HOMEOBOX7. Analysis of the predicted transcription factor regulatory networks for these genes suggested that a complex interplay of common and tissue-speciﬁc components contributes to the coordination of post-recovery responses to stress in woody plants.
Aquaporins are membrane channels that facilitate water movement across cell membranes. In plants, aquaporins contribute to water relations. Here, we establish a new link between aquaporin-dependent tissue hydraulics and auxin-regulated root development in Arabidopsis thaliana. We report that most aquaporin genes are repressed during lateral root formation and by exogenous auxin treatment. Auxin reduces root hydraulic conductivity both at the cell and whole-organ levels. The highly expressed aquaporin PIP2;1 is progressively excluded from the site of the auxin response maximum in lateral root primordia (LRP) whilst being maintained at their base and underlying vascular tissues. Modelling predicts that the positive and negative perturbations of PIP2;1 expression alter water ﬂow into LRP, thereby slowing lateral root emergence (LRE). Consistent with this mechanism, pip2;1 mutants and PIP2;1-overexpressing lines exhibit delayed LRE. We conclude that auxin promotes LRE by regulating the spatial and temporal distribution of aquaporin-dependent root tissue water transport.