Glutamine induces lateral root initiation, stress responses, and disease resistance in Arabidopsis.

The production of glutamine (Gln) from NO3- and NH4+ requires ATP, reducing power, and carbon skeletons. Plants may redirect these resources to other physiological processes using Gln directly. However, feeding Gln as the sole nitrogen (N) source has complex effects on plants. Under optimal concentrations, Arabidopsis (Arabidopsis thaliana) seedlings grown on Gln have similar primary root lengths, more lateral roots, smaller leaves, and higher amounts of amino acids and proteins compared to those grown on NH4NO3. While high levels of Gln accumulate in Arabidopsis seedlings grown on Gln, the expression of GLUTAMINE SYNTHETASE1; 1 (GLN1; 1), GLN1; 2, and GLN1; 3 encoding cytosolic GS1 increases and expression of GLN2 encoding chloroplastic GS2 decreases. These results suggest that Gln has distinct effects on regulating GLN1 and GLN2 gene expression. Notably, Arabidopsis seedlings grown on Gln have an unexpected gene expression profile. Compared with NH4NO3, which activates growth-promoting genes, Gln preferentially induces stress- and defense-responsive genes. Consistent with the gene expression data, exogenous treatment with Gln enhances disease resistance in Arabidopsis. The induction of Gln-responsive genes, including PATHOGENESIS-RELATED1, SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1, WRKY54, and WALL ASSOCIATED KINASE1, is compromised in salicylic acid (SA) biosynthetic and signaling mutants under Gln treatments. Together, these results suggest that Gln may partly interact with the SA pathway to trigger plant immunity.

Glutamine Metabolism, Sensing and Signaling in Plants.

Glutamine (Gln) is the first amino acid synthesized in nitrogen (N) assimilation in plants. Gln synthetase (GS), converting glutamate (Glu) and NH4+ into Gln at the expense of ATP, is one of the oldest enzymes in all life domains. Plants have multiple GS isoenzymes that work individually or cooperatively to ensure that the Gln supply is sufficient for plant growth and development under various conditions. Gln is a building block for protein synthesis and an N-donor for the biosynthesis of amino acids, nucleic acids, amino sugars and vitamin B coenzymes. Most reactions using Gln as an N-donor are catalyzed by Gln amidotransferase (GAT) that hydrolyzes Gln to Glu and transfers the amido group of Gln to an acceptor substrate. Several GAT domain-containing proteins of unknown function in the reference plant Arabidopsis thaliana suggest that some metabolic fates of Gln have yet to be identified in plants. In addition to metabolism, Gln signaling has emerged in recent years. The N regulatory protein PII senses Gln to regulate arginine biosynthesis in plants. Gln promotes somatic embryogenesis and shoot organogenesis with unknown mechanisms. Exogenous Gln has been implicated in activating stress and defense responses in plants. Likely, Gln signaling is responsible for some of the new Gln functions in plants.

Arabidopsis ACT DOMAIN REPEAT9 represses glucose signaling pathways.

Nutrient sensing and signaling are critical for plants to coordinate growth and development in response to nutrient availability. Plant ACT DOMAIN REPEAT (ACR) proteins have been proposed to serve as nutrient sensors, but their functions remain largely unknown. Here, we showed that Arabidopsis (Arabidopsis thaliana) ACR9 might function as a repressor in glucose (Glc) signaling pathways. ACR9 was highly expressed in the leaves, and its expression was downregulated by sugars. Interestingly, the acr9-1 and acr9-2 T-DNA insertion mutants were hypersensitive to Glc during seedling growth, development, and anthocyanin accumulation. Nitrogen deficiency increased the mutants' sensitivity to Glc. The expression of sugar-responsive genes was also significantly enhanced in the acr9 mutants. By contrast, the 35S:ACR9 and 35S:ACR9-GFP overexpression (OE) lines were insensitive to Glc during early seedling development. The Glc signaling pathway is known to interact with the plant hormone abscisic acid (ABA). Notably, the acr9 mutants were also hypersensitive to ABA during early seedling development. The Glc sensor HEXOKINASE1 (HXK1) and the energy sensor SUCROSE NON-FERMENTING1 (SNF1)-RELATED PROTEIN KINASE1 (SnRK1) are key components of the Glc signaling pathways. The acr9-1/hxk1-3 and acr9-1/snrk1 double mutants were no longer hypersensitive to Glc, indicating that functional HXK1 and SnRK1 were required for the acr9-1 mutant to be hypersensitive to Glc. Together, these results suggest that ACR9 is a repressor of the Glc signaling pathway, which may act independently or upstream of the HXK1-SnRK1 signaling module.

THIAMIN REQUIRING2 is involved in thiamin diphosphate biosynthesis and homeostasis.

The THIAMIN REQUIRING2 (TH2) protein comprising a mitochondrial targeting peptide followed by a transcription enhancement A and a haloacid dehalogenase domain is a thiamin monophosphate (TMP) phosphatase in the vitamin B1 biosynthetic pathway. The Arabidopsis th2-3 T-DNA insertion mutant was chlorotic and deficient in thiamin diphosphate (TDP). Complementation assays confirmed that haloacid dehalogenase domain alone was sufficient to rescue the th2-3 mutant. In pTH2:TH2-GFP/th2-3 complemented plants, the TH2-GFP was localized to the cytosol, mitochondrion, and nucleus, indicating that the vitamin B1 biosynthetic pathway extended across multi-subcellular compartments. Engineered TH2-GFP localized to the cytosol, mitochondrion, nucleus, and chloroplast, could complement the th2 mutant. Together, these results highlight the importance of intracellular TMP and thiamin trafficking in vitamin B1 biosynthesis. In an attempt to enhance the production of thiamin, we created various constructs to overexpress TH2-GFP in the cytosol, mitochondrion, chloroplast, and nucleus. Unexpectedly, overexpressing TH2-GFP resulted in an increase rather than a decrease in TMP. While studies on th2 mutants support TH2 as a TMP phosphatase, analyses of TH2-GFP overexpression lines implicating TH2 may also function as a TDP phosphatase in planta. We propose a working model that the TMP/TDP phosphatase activity of TH2 connects TMP, thiamin, and TDP into a metabolic cycle. The TMP phosphatase activity of TH2 is required for TDP biosynthesis, and the TDP phosphatase activity of TH2 may modulate TDP homeostasis in Arabidopsis.

Glutamate: A multifunctional amino acid in plants.

Glutamate (Glu) is a versatile metabolite and a signaling molecule in plants. Glu biosynthesis is associated with the primary nitrogen assimilation pathway. The conversion between Glu and 2-oxoglutarate connects Glu metabolism to the tricarboxylic acid cycle, carbon metabolism, and energy production. Glu is the predominant amino donor for transamination reactions in the cell. In addition to protein synthesis, Glu is a building block for tetrapyrroles, glutathione, and folate. Glu is the precursor of γ-aminobutyric acid that plays an important role in balancing carbon/nitrogen metabolism and various cellular processes. Glu can conjugate to the major auxin indole 3-acetic acid (IAA), and IAA-Glu is destined for oxidative degradation. Glu also conjugates with isochorismate for the production of salicylic acid. Accumulating evidence indicates that Glu functions as a signaling molecule to regulate plant growth, development, and defense responses. The ligand-gated Glu receptor-like proteins (GLRs) mediate some of these responses. However, many of the Glu signaling events are GLR-independent. The receptor perceiving extracellular Glu as a danger signal is still unknown. In addition to GLRs, Glu may act on receptor-like kinases or receptor-like proteins to trigger immune responses. Glu metabolism and Glu signaling may entwine to regulate growth, development, and defense responses in plants.

Nitrogen deficiency- and sucrose-induced anthocyanin biosynthesis is modulated by HISTONE DEACETYLASE15 in Arabidopsis.

Anthocyanin accumulation is a hallmark response to nitrogen (N) deficiency in Arabidopsis. Although the regulation of anthocyanin biosynthesis has been extensively studied, the roles of chromatin modification in this process are largely unknown. In this study we show that anthocyanin accumulation induced by N deficiency is modulated by HISTONE DEACETYLASE15 (HDA15) in Arabidopsis seedlings. The hda15-1 T-DNA insertion mutant accumulated more anthocyanins than the wild-type when the N supply was limited, and this was caused by up-regulation of anthocyanin biosynthetic and regulatory genes in the mutant. The up-regulated genes also had increased levels of histone acetylation in the mutant. The accumulation of anthocyanins induced by sucrose and methyl jasmonate, but not that induced by H2O2 and phosphate starvation, was also greater in the hda15-1 mutant. While sucrose increased histone acetylation in the hda15-1 mutant in genes in a similar manner to that caused by N deficiency, methyl jasmonate only enhanced histone acetylation in the genes involved in anthocyanin biosynthesis. Our results suggest that different stresses act through distinct regulatory modules to activate anthocyanin biosynthesis, and that HDA15-mediated histone modification modulates the expression of anthocyanin biosynthetic and regulatory genes to avoid overaccumulation in response to N deficiency and other stresses.

The lineage and diversity of putative amino acid sensor ACR proteins in plants.

Amino acid metabolic enzymes often contain a regulatory ACT domain, named for aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase). Arabidopsis encodes 12 putative amino acid sensor ACT repeat (ACR) proteins, all containing ACT repeats but no identifiable catalytic domain. Arabidopsis ACRs comprise three groups based on domain composition and sequence: group I and II ACRs contain four ACTs each, and group III ACRs contain two ACTs. Previously, all three groups had been documented only in Arabidopsis. Here, we extended this to algae and land plants, showing that all three groups of ACRs are present in most, if not all, land plants, whereas among algal ACRs, although quite diverse, only group III is conserved. The appearance of canonical group I and II ACRs thus accompanied the evolution of plants from living in water to living on land. Alignment of ACTs from plant ACRs revealed a conserved motif, DRPGLL, at the putative ligand-binding site. Notably, the unique features of the DRPGLL motifs in each ACT domain are conserved in ACRs from algae to land plants. The conservation of plant ACRs is reminiscent of that of human cellular arginine sensor for mTORC1 (CASTOR1), a member of a small protein family highly conserved in animals. CASTOR proteins also have four ACT domains, although the sequence identities between ACRs and CASTORs are very low. Thus, plant ACRs and animal CASTORs may have adapted the regulatory ACT domains from a more ancient metabolic enzyme, and then evolved independently.