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 thalianaUGT 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.

Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PAL1 and PAL2. However, a complete blocking of flavonoid biosynthesis at CHS released PAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols.

Summary: Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis‐glycoside is correlated with shorter plant stature and reduced PAT. Specific flavonoid‐biosynthetic or flavonoid‐glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high‐performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [³H]‐indole‐3‐acetic acid tracer. The flavonoid 3‐O‐glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2‐dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3‐O‐rhamnoside‐7‐O‐rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport. These results indicate that kaempferol 3‐O‐rhamnoside‐7‐O‐rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.

Several factors regulate plant organ growth polarity. tortifolia2 (tor2), a right-handed helical growth mutant, has a conservative replacement of Arg-2 with Lys in the α-tubulin 4 protein. Based on a published high-resolution (2.89 Å) tubulin structure, we predict that Arg-2 of α-tubulin forms hydrogen bonds with the GTPase domain of β-tubulin, and structural modeling suggests that these contacts are interrupted in tor2. Consistent with this, we found that microtubule dynamicity is reduced in the tor2 background. We investigated the developmental origin of the helical growth phenotype using tor2. One hypothesis predicts that cell division patterns cause helical organ growth in Arabidopsis thaliana mutants. However, cell division patterns of tor2 root tips appear normal. Experimental uncoupling of cell division and expansion suggests that helical organ growth is based on cell elongation defects only. Another hypothesis is that twisting is due to inequalities in expansion of epidermal and cortical tissues. However, freely growing leaf trichomes of tor2 mutants show right-handed twisting and cortical microtubules form left-handed helices as early as the unbranched stage of trichome development. Trichome twisting is inverted in double mutants with tor3, a left-handed mutant. Single tor2 suspension cells also exhibit handed twisting. Thus, twisting of tor2 mutant organs appears to be a higher-order expression of the helical expansion of individual cells.

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

In this study, genome-wide expression profiling based on Affymetrix ATH1 arrays was used to identify discriminating responses of Arabidopsis thaliana to five herbicides, which contain active ingredients targeting two different branches of amino acid biosynthesis. One herbicide contained glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), while the other four herbicides contain different acetolactate synthase (ALS) inhibiting compounds. In contrast to the herbicide containing glyphosate, which affected only a few transcripts, many effects of the ALS inhibiting herbicides were revealed based on transcriptional changes related to ribosome biogenesis and translation, secondary metabolism, cell wall modification and growth. The expression pattern of a set of 101 genes provided a specific, composite signature that was distinct from other major stress responses and differentiated among herbicides targeting the same enzyme (ALS) or containing the same chemical class of active ingredient (sulfonylurea). A set of homologous genes could be identified in Brassica napus that exhibited a similar expression pattern and correctly distinguished exposure to the five herbicides. Our results show the ability of a limited number of genes to classify and differentiate responses to closely related herbicides in A. thaliana and B. napus and the transferability of a complex transcriptional signature across species.