Stephan Pollmann

Auxin transport through PIN-FORMED 3 (PIN3) controls shade avoidance and fitness during competition

Plants grow in dense vegetations at the risk of being out-competed by neighbors. To increase their competitive power, plants display adaptive responses, such as rapid shoot elongation (shade avoidance) to consolidate light capture. These responses are induced upon detection of proximate neighbors through perception of the reduced ratio between red (R) and far-red (FR) light that is typical for dense vegetations. The plant hormone auxin is a central regulator of plant development and plasticity, but until now it has been unknown how auxin transport is controlled to regulate shade-avoidance responses. Here, we show that low R:FR detection changes the cellular location of the PIN-FORMED 3 (PIN3) protein, a regulator of auxin efflux, in Arabidopsis seedlings. As a result, auxin levels in the elongating hypocotyls are increased under low R:FR. Seedlings of the pin3-3 mutant lack this low R:FR-induced increase of endogenous auxin in the hypocotyl and, accordingly, have no elongation response to low R:FR. We hypothesize that low R:FR-induced stimulation of auxin biosynthesis drives the regulation of PIN3, thus allowing shade avoidance to occur. The adaptive significance of PIN3-mediated control of shade-avoidance is shown in plant competition studies. It was found that pin3 mutants are outcompeted by wild-type neighbors who suppress fitness of pin3-3 by 40%. We conclude that low R:FR modulates the auxin distribution by a change in the cellular location of PIN3, and that this control can be of great importance for plants growing in dense vegetations.

Characterization of TCTP, the Translationally Controlled Tumor Protein, from Arabidopsis thaliana

The translationally controlled tumor protein (TCTP) is an important component of the TOR (target of rapamycin) signaling pathway, the major regulator of cell growth in animals and fungi. TCTP acts as the guanine nucleotide exchange factor of the Ras GTPase Rheb that controls TOR activity in Drosophila melanogaster. We therefore examined the role of Arabidopsis thaliana TCTP in planta. Plant TCTPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. Green fluorescent protein reporter lines show that Arabidopsis TCTP is expressed throughout plant tissues and developmental stages with increased expression in meristematic and expanding cells. Knockout of TCTP leads to a male gametophytic phenotype with normal pollen formation and germination but impaired pollen tube growth. Silencing of TCTP by RNA interference slows vegetative growth; leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCTP as an important regulator of growth in plants and imply a function of plant TCTP as a mediator of TOR activity similar to that known in nonplant systems.

ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis

Auxin is a key coordinative signal required for many aspects of plant development and its levels are controlled by auxin metabolism and intercellular auxin transport. Here we find that a member of PIN auxin transporter family, PIN8 is expressed in male gametophyte of Arabidopsis thaliana and has a crucial role in pollen development and functionality. Ectopic expression in sporophytic tissues establishes a role of PIN8 in regulating auxin homoeostasis and metabolism. PIN8 co-localizes with PIN5 to the endoplasmic reticulum (ER) where it acts as an auxin transporter. Genetic analyses reveal an antagonistic action of PIN5 and PIN8 in the regulation of intracellular auxin homoeostasis and gametophyte as well as sporophyte development. Our results reveal a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters regulate cellular auxin homoeostasis and maintain the auxin levels optimal for pollen development and pollen tube growth.

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.

Plant oxylipins: Plant responses to 12-oxo-phytodienoic acid are governed by its specific structural and functional properties

One of the most challenging questions in modern plant science is how plants regulate their morphological and developmental adaptation in response to changes in their biotic and abiotic environment. A comprehensive elucidation of the underlying mechanisms will help shed light on the extremely efficient strategies of plants in terms of survival and propagation. In recent years, a number of environmental stress conditions have been described as being mediated by signaling molecules of the oxylipin family. In this context, jasmonic acid, its biosynthetic precursor, 12‐oxo‐phytodienoic acid (OPDA), and also reactive electrophilic species such as phytoprostanes play pivotal roles. Although our understanding of jasmonic acid‐dependent processes and jasmonic acid signal‐transduction cascades has made considerable progress in recent years, knowledge of the regulation and mode of action of OPDA‐dependent plant responses is just emerging. This minireview focuses on recent work concerned with the elucidation of OPDA‐specific processes in plants. In this context, aspects such as the differential recruitment of OPDA, either by de novo biosynthesis or by release from cyclo‐oxylipin‐galactolipids, and the conjugation of free OPDA are discussed.

The jasmonic acid signaling pathway is linked to auxin homeostasis through the modulation of YUCCA8 and YUCCA9 gene expression

Interactions between phytohormones play important roles in the regulation of plant growth and development, but knowledge of the networks controlling hormonal relationships, such as between oxylipins and auxins, is just emerging. Here, we report the transcriptional regulation of two Arabidopsis YUCCA genes, YUC8 and YUC9, by oxylipins. Similar to previously characterized YUCCA family members, we show that both YUC8 and YUC9 are involved in auxin biosynthesis, as demonstrated by the increased auxin contents and auxin-dependent phenotypes displayed by gain-of-function mutants as well as the significantly decreased indole-3-acetic acid (IAA) levels in yuc8 and yuc8/9 knockout lines. Gene expression data obtained by qPCR analysis and microscopic examination of promoter-reporter lines reveal an oxylipin-mediated regulation of YUC9 expression that is dependent on the COI1 signal transduction pathway. In support of these findings, the roots of the analyzed yuc knockout mutants displayed a reduced response to methyl jasmonate (MeJA). The similar response of the yuc8 and yuc9 mutants to MeJA in cotyledons and hypocotyls suggests functional overlap of YUC8 and YUC9 in aerial tissues, while their function in roots shows some specificity, probably in part related to different spatio-temporal expression patterns of the two genes. These results provide evidence for an intimate functional relationship between oxylipin signaling and auxin homeostasis.

Identification of an ABCB/P-glycoprotein-specific inhibitor of auxin transport by chemical genomics.

Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC50 value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.

Regulation of ABCB1/PGP1‐catalysed auxin transport by linker phosphorylation

Polar transport of the plant hormone auxin is controlled by PIN‐ and ABCB/PGP‐efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co‐immunoprecipitation (co‐IP) and shotgun LC–MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In‐vitro and yeast expression analyses indicated that PID specifically modulates ABCB1‐mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co‐transfection in tobacco revealed that PID enhances ABCB1‐mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho‐proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter‐active impact on ABCB1 activity that is coordinated by TWD1–PID interaction.

Endophytic root colonization of gramineous plants by Herbaspirillum frisingense

Herbaspirillum frisingense is a diazotrophic betaproteobacterium isolated from C4-energy plants, for example Miscanthus sinensis. To demonstrate endophytic colonization unequivocally, immunological labeling techniques using monospecific polyclonal antibodies against two H. frisingense strains and green fluorescent protein (GFP)-fluorescence tagging were applied. The polyclonal antibodies enabled specific in situ identification and very detailed localization of H. frisingense isolates Mb11 and GSF30(T) within roots of Miscanthusxgiganteus seedlings. Three days after inoculation, cells were found inside root cortex cells and after 7 days they were colonizing the vascular tissue in the central cylinder. GFP-tagged H. frisingense strains could be detected and localized in uncut root material by confocal laser scanning microscopy and were found as endophytes in cortex cells, intercellular spaces and the central cylinder of barley roots. Concerning the production of potential plant effector molecules, H. frisingense strain GSF30(T) tested positive for the production of indole-3-acetic acid, while Mb11 was shown to produce N-acylhomoserine lactones, and both strains were able to utilize 1-aminocyclopropane-1-carboxylate (ACC), providing an indication of the activity of an ACC-deaminase. These results clearly present H. frisingense as a true plant endophyte and, although initial greenhouse experiments did not lead to clear plant growth stimulation, demonstrate the potential of this species for beneficial effects on the growth of crop plants.

DONGLE and DEFECTIVE IN ANTHER DEHISCENCE1 Lipases Are Not Essential for Wound- and Pathogen-Induced Jasmonate Biosynthesis: Redundant Lipases Contribute to Jasmonate Formation

Lipases are involved in the generation of jasmonates, which regulate responses to biotic and abiotic stresses. Two sn-1-specific acyl hydrolases, DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) and DONGLE (DGL), have been reported to be localized in plastids and to be essential and sufficient for jasmonate biosynthesis in Arabidopsis (Arabidopsis thaliana) leaves. Here, we show that levels of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid in three different DGL RNA interference lines and the dad1 mutant were similar to wild-type levels during the early wound response as well as after Pseudomonas infection. Due to the lack of sn-2 substrate specificity, synthesis of dinor OPDA was not expected and also not found to be affected in DGL knockdown and DGL-overexpressing lines. As reported, DAD1 participates in jasmonate formation only in the late wound response. In addition, DGL protein was found to be localized in lipid bodies and not in plastids. Furthermore, jasmonate levels in 16 additional mutants defective in the expression of lipases with predicted chloroplast localization did not show strong differences from wild-type levels after wounding, except for a phospholipase A (PLA) PLA-Iγ1 (At1g06800) mutant line that displayed diminished wound-induced dinor OPDA, OPDA, and jasmonic acid levels. A quadruple mutant defective in four DAD1-like lipases displayed similar jasmonate levels as the mutant line of PLA-Iγ1 after wounding. Hence, we identify PLA-Iγ1 as a novel target gene to manipulate jasmonate biosynthesis. Our results suggest that, in addition to DAD1 and PLA-Iγ1, still unidentified enzymes with sn-1 and sn-2 hydrolase activity are involved in wound- and pathogen-induced jasmonate formation, indicating functional redundancy within the lipase family.