SA and NHP glucosyltransferase UGT76B1 affects plant defense in both SID2- and NPR1-dependent and independent manner.

KEY MESSAGE: The small-molecule glucosyltransferase loss-of-function mutant ugt76b1 exhibits both SID2- or NPR1-dependent and independent facets of enhanced plant immunity, whereupon FMO1 is required for the SID2 and NPR1 independence. The small-molecule glucosyltransferase UGT76B1 inactivates salicylic acid (SA), isoleucic acid (ILA), and N-hydroxypipecolic acid (NHP). ugt76b1 loss-of-function plants manifest an enhanced defense status. Thus, we were interested how UGT76B1 genetically integrates in defense pathways and whether all impacts depend on SA and NHP. We study the integration of UGT76B1 by transcriptome analyses of ugt76b1. The comparison of transcripts altered by the loss of UGT76B1 with public transcriptome data reveals both SA-responsive, ISOCHORISMATE SYNTHASE 1/SALICYLIC ACID INDUCTION DEFICIENT 2 (ICS1/SID2)- and NON EXPRESSOR OF PR GENES 1 (NPR1)-dependent, consistent with the role of UGT76B1 in glucosylating SA, and SA-non-responsive, SID2/NPR1-independent genes. We also discovered that UGT76B1 impacts on a group of genes showing non-SA-responsiveness and regulation by infections independent from SID2/NPR1. Enhanced resistance of ugt76b1 against Pseudomonas syringae is partially independent from SID2 and NPR1. In contrast, the ugt76b1-activated resistance is completely dependent on FMO1 encoding the NHP-synthesizing FLAVIN-DEPENDENT MONOOXYGENASE 1). Moreover, FMO1 ranks top among the ugt76b1-induced SID2- and NPR1-independent pathogen responsive genes, suggesting that FMO1 determines the SID2- and NPR1-independent effect of ugt76b1. Furthermore, the genetic study revealed that FMO1, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), SID2, and NPR1 are required for the SA-JA crosstalk and senescence development of ugt76b1, indicating that EDS1 and FMO1 have a similar effect like stress-induced SA biosynthesis (SID2) or the key SA signaling regulator NPR1. Thus, UGT76B1 influences both SID2/NPR1-dependent and independent plant immunity, and the SID2/NPR1 independence is relying on FMO1 and its product NHP, another substrate of UGT76B1.

UGT76B1, a promiscuous hub of small molecule-based immune signaling, glucosylates N-hydroxypipecolic acid, and balances plant immunity.

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-ß-glucoside and NHP glucose ester, whereupon only NHP-O-ß-glucoside formation requires a functional SA pathway. The ugt76b1 mutants specifically fail to generate the NHP-O-ß-glucoside, and recombinant UGT76B1 synthesizes NHP-O-ß-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-ß-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.

Identification and functional characterization of two bamboo FD gene homologs having contrasting effects on shoot growth and flowering.

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.

Corrigendum: The Defense-Related Isoleucic Acid Differentially Accumulates in Arabidopsis Among Branched-Chain Amino Acid-Related 2-Hydroxy Carboxylic Acids.

[This corrects the article DOI: 10.3389/fpls.2018.00766.].

The isoleucic acid triad: distinct impacts on plant defense, root growth, and formation of reactive oxygen species.

Isoleucic acid (ILA), a branched-chain amino acid-related 2-hydroxycarboxylic acid, occurs ubiquitously in plants. It enhances pathogen resistance and inhibits root growth of Arabidopsis. The salicylic acid (SA) glucosyltransferase UGT76B1 is able to conjugate ILA. Here, we investigate the role of ILA in planta in Arabidopsis and reveal a triad of distinct responses to this small molecule. ILA synergistically co-operates with SA to activate SA-responsive gene expression and resistance in a UGT76B1-dependent manner in agreement with the observed competitive ILA-dependent repression of SA glucosylation by UGT76B1. However, ILA also shows an SA-independent stress response. Nitroblue tetrazolium staining and pharmacological experiments indicate that ILA induces superoxide formation of the wild type and of an SA-deficient (NahG sid2) line. In contrast, the inhibitory effect of ILA on root growth is independent of both SA and superoxide induction. These effects of ILA are specific and distinct from its isomeric compound leucic acid and from the amino acid isoleucine. Leucic acid and isoleucine do not induce expression of defense marker genes or superoxide production, whereas both compounds inhibit root growth. All three responses to ILA are also observed in Brassica napus.

The Defense-Related Isoleucic Acid Differentially Accumulates in Arabidopsis Among Branched-Chain Amino Acid-Related 2-Hydroxy Carboxylic Acids.

The branched-chain amino acid (BCAA) related 2-hydroxy carboxylic acid isoleucic acid (ILA) enhances salicylic acid-mediated pathogen defense in Arabidopsis thaliana. ILA has been identified in A. thaliana as its glucose conjugate correlated with the activity of the small-molecule glucosyltransferase UGT76B1, which can glucosylate both salicylic acid and ILA in vitro. However, endogenous levels of the ILA aglycon have not yet been determined in planta. To quantify ILA as well as the related leucic acid (LA) and valic acid (VA) in plant extracts, a sensitive method based on the derivatization of small carboxylic acids by silylation and gas chromatography-mass spectrometric analysis was developed. ILA was present in all species tested including several monocotyledonous and dicotyledonous plants as well as broadleaf and coniferous trees, whereas LA and VA were only detectable in a few species. In A. thaliana both ILA and LA were found. However, their levels varied during plant growth and in root vs. leaves. ILA levels were higher in 2-week-old leaves and decreased in older plants, whereas LA exhibited a reverted accumulation pattern. Roots displayed higher ILA and LA levels compared to leaves. ILA was inversely related to UGT76B1 expression level indicating that UGT76B1 glucosylates ILA in planta. In contrast, LA was not affected by the expression of UGT76B1. To address the relation of both 2-hydroxy acids to plant defense, we studied ILA and LA levels upon infection by Pseudomonas syringae. LA abundance remained unaffected, whereas ILA was reduced. This change suggests an ILA-related attenuation of the salicylic acid response. Collectively, the BCAA-related ILA and LA differentially accumulated in Arabidopsis, supporting a specific role and regulation of the defense-modulating small-molecule ILA among these 2-hydroxy acids. The new sensitive method will pave the way to further unravel their role in plants.

The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence.

Plants coordinate and tightly regulate pathogen defense by the mostly antagonistic salicylate (SA)- and jasmonate (JA)-mediated signaling pathways. Here, we show that the previously uncharacterized glucosyltransferase UGT76B1 is a novel player in this SA-JA signaling crosstalk. UGT76B1 was selected as the top stress-induced isoform among all 122 members of the Arabidopsis thaliana UGT family. Loss of UGT76B1 function leads to enhanced resistance to the biotrophic pathogen Pseudomonas syringae and accelerated senescence but increased susceptibility toward necrotrophic Alternaria brassicicola. This is accompanied by constitutively elevated SA levels and SA-related marker gene expression, whereas JA-dependent markers are repressed. Conversely, UGT76B1 overexpression has the opposite effect. Thus, UGT76B1 attenuates SA-dependent plant defense in the absence of infection, promotes the JA response, and delays senescence. The ugt76b1 phenotypes were SA dependent, whereas UGT76B1 overexpression indicated that this gene possibly also has a direct effect on the JA pathway. Nontargeted metabolomic analysis of UGT76B1 knockout and overexpression lines using ultra-high-resolution mass spectrometry and activity assays with the recombinant enzyme led to the ab initio identification of isoleucic acid (2-hydroxy-3-methyl-pentanoic acid) as a substrate of UGT76B1. Exogenously applied isoleucic acid increased resistance against P. syringae infection. These findings indicate a novel link between amino acid-related molecules and plant defense that is mediated by small-molecule glucosylation.

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts.

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.

Detection of the activating JAK2 V617F mutation in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative diseases.

The discovery of the activating V617F mutation in the JAK2 tyrosine kinase in a high proportion of patients with Ph- chronic myeloproliferative diseases (CMPD) represents a diagnostic breakthrough for these disorders. Trephine bone marrow biopsy is an essential part of the diagnostic workup of CMPD and represents a valuable archival source of DNA. Therefore, we studied 152 paraffin-embedded trephines with CMPD and related disorders for the presence of the V617F mutation, using both allele-specific polymerase chain reaction (PCR) and nested PCR with subsequent digestion with BsaXI. Only 6 of 152 (4%) samples were not evaluable because of poor DNA quality. The V617F mutation was detected in 27 of 28 (96%) cases of polycythemia vera, 17 of 23 (74%) cases of essential thrombocythemia, 28 of 45 (62%) cases of chronic idiopathic myelofibrosis, six of eight (75%) cases of CMPD unclassified, and two of four (50%) cases of myelodysplastic/myeloproliferative syndrome. Ph+ chronic myelogenous leukemia (four cases), reactive (secondary) erythrocytosis (14 cases), and thrombocytosis (one case) as well as normal controls (19 cases) all lacked the V617F mutation. Based on results of BsaXI digestion and sequencing, 24 of 54 (44%) evaluable V617F+ cases were considered homozygously mutated. Thus, detection of the V617F JAK2 mutation is feasible in paraffin-embedded trephine biopsies and represents a major advance in the diagnostic evaluation of CMPD.

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism.

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato . The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.