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.

Ectopic expression of a bacterial thiamin monophosphate kinase enhances vitamin B1 biosynthesis in plants.

Plants and bacteria have distinct pathways to synthesize the bioactive vitamin B1 thiamin diphosphate (TDP). In plants, thiamin monophosphate (TMP) synthesized in the TDP biosynthetic pathway is first converted to thiamin by a phosphatase, which is then pyrophosphorylated to TDP. In contrast, bacteria use a TMP kinase encoded by ThiL to phosphorylate TMP to TDP directly. The Arabidopsis THIAMIN REQUIRING2 (TH2)-encoded phosphatase is involved in TDP biosynthesis. The chlorotic th2 mutants have high TMP and low thiamin and TDP. Ectopic expression of Escherichia coli ThiL and ThiL-GFP rescued the th2-3 mutant, suggesting that the bacterial TMP kinase could directly convert TMP into TDP in Arabidopsis. These results provide direct evidence that the chlorotic phenotype of th2-3 is caused by TDP rather than thiamin deficiency. Transgenic Arabidopsis harboring engineered ThiL-GFP targeting to the cytosol, chloroplast, mitochondrion, or nucleus accumulated higher TDP than the wild type (WT). Ectopic expression of E. coli ThiL driven by the UBIQUITIN (UBI) promoter or an endosperm-specific GLUTELIN1 (GT1) promoter also enhanced TDP biosynthesis in rice. The pUBI:ThiL transgenic rice accumulated more TDP and total vitamin B1 in the leaves, and the pGT1:ThiL transgenic lines had higher TDP and total vitamin B1 in the seeds than the WT. Total vitamin B1 only increased by approximately 25-30% in the polished and unpolished seeds of the pGT1:ThiL transgenic rice compared to the WT. Nevertheless, these results suggest that genetic engineering of a bacterial vitamin B1 biosynthetic gene downstream of TMP can enhance vitamin B1 production in rice.

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.

Transformation of nad7 into the nuclear genome rescues the slow growth3 mutant in Arabidopsis.

Plant pentatricopeptide repeat (PPR) proteins are mostly involved in chloroplast or mitochondrial RNA metabolism. However, direct evidence that correction of the molecular defects in the organelles can restore the plant phenotypes has yet to be demonstrated in a ppr mutant. Arabidopsis slow growth3 (slo3), a ppr mutant, is impaired in the splicing of mitochondrial nad7 intron 2. Here, we have used slo3 as an example to demonstrate that transformation of correctly spliced nad7 into the nuclear genome and targeting the Nad7 subunit into mitochondria can restore complex I activity and plant phenotypes in the mutant. These results provide direct evidence that the strong growth and developmental phenotypes of the slo3 mutant are caused by defects in mitochondrial nad7.

Early molecular events associated with nitrogen deficiency in rice seedling roots.

Nitrogen (N) deficiency is one of the most common problems in rice. The symptoms of N deficiency are well documented, but the underlying molecular mechanisms are largely unknown in rice. Here, we studied the early molecular events associated with N starvation (-N, 1 h), focusing on amino acid analysis and identification of -N-regulated genes in rice roots. Interestingly, levels of glutamine rapidly decreased within 15 min of -N treatment, indicating that part of the N-deficient signals could be mediated by glutamine. Transcriptome analysis revealed that genes involved in metabolism, plant hormone signal transduction (e.g. abscisic acid, auxin, and jasmonate), transporter activity, and oxidative stress responses were rapidly regulated by -N. Some of the -N-regulated genes encode transcription factors, protein kinases and protein phosphatases, which may be involved in the regulation of early -N responses in rice roots. Previously, we used similar approaches to identify glutamine-, glutamate-, and ammonium nitrate-responsive genes. Comparisons of the genes induced by different forms of N with the -N-regulated genes identified here have provided a catalog of potential N regulatory genes for further dissection of the N signaling pathwys in rice.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis.

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Identification of early ammonium nitrate-responsive genes in rice roots.

Ammonium has long been used as the predominant form of nitrogen source for paddy rice (Oryza sativa). Recently, increasing evidence suggests that nitrate also plays an important role for nitrogen acquisition in the rhizosphere of waterlogged paddy rice. Ammonium and nitrate have a synergistic effect on promoting rice growth. However, the molecular responses induced by simultaneous treatment with ammonium and nitrate have been less studied in rice. Here, we performed transcriptome analysis to identify genes that are rapidly regulated by ammonium nitrate (1.43 mM, 30 min) in rice roots. The combination of ammonium and nitrate preferentially induced the expression of nitrate-responsive genes. Gene ontology enrichment analysis revealed that the early ammonium nitrate-responsive genes were enriched in "regulation of transcription, DNA-dependent" and "protein amino acid phosphorylation" indicating that some of the genes identified in this study may play an important role in nitrogen sensing and signaling. Several defense/stress-responsive genes, including some encoding transcription factors and mitogen-activated protein kinase kinase kinases, were also rapidly induced by ammonium nitrate. These results suggest that nitrogen metabolism, signaling, and defense/stress responses are interconnected. Some of the genes identified here may be involved in the interaction of nitrogen signaling and defense/stress-response pathways in plants.

The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

Thiamin diphosphate (TPP, vitamin B1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes.

Exogenous glutamate rapidly induces the expression of genes involved in metabolism and defense responses in rice roots.

BACKGROUND: Glutamate is an active amino acid. In addition to protein synthesis and metabolism, increasing evidence indicates that glutamate may also function as a signaling molecule in plants. Still, little is known about the nutritional role of glutamate and genes that are directly regulated by glutamate in rice. RESULTS: Exogenous glutamate could serve as a nitrogen nutrient to support the growth of rice seedlings, but it was not as effective as ammonium nitrate or glutamine. In nitrogen-starved rice seedlings, glutamate was the most abundant free amino acid and feeding of glutamate rapidly and significantly increased the endogenous levels of glutamine, but not glutamate. These results indicated that glutamate was quickly metabolized and converted to the other nitrogen-containing compounds in rice. Transcriptome analysis revealed that at least 122 genes involved in metabolism, transport, signal transduction, and stress responses in the roots were rapidly induced by 2.5 mM glutamate within 30 min. Many of these genes were also up-regulated by glutamine and ammonium nitrate. Still, we were able to identify some transcription factor, kinase/phosphatase, and elicitor-responsive genes that were specifically or preferentially induced by glutamate. CONCLUSIONS: Glutamate is a functional amino acid that plays important roles in plant nutrition, metabolism, and signal transduction. The rapid and specific induction of transcription factor, kinase/phosphatase and elicitor-responsive genes suggests that glutamate may efficiently amplify its signal and interact with other signaling pathways to regulate metabolism, growth and defense responses in rice.

Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing.

The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg(300)-to-Trp(300) and Pro(313)-to-Ser(313) amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles.

Dysfunctional chloroplasts up-regulate the expression of mitochondrial genes in Arabidopsis seedlings.

Chloroplasts and mitochondria play important roles in maintaining metabolic and energy homeostasis in the plant cell. The interactions between these two organelles, especially photosynthesis and respiration, have been intensively studied. Still, little is known about the regulation of mitochondrial gene expression by chloroplasts and vice versa. The gene expression machineries in chloroplasts and mitochondria rely heavily on the nuclear genome. Thus, the interactions between nucleus and these organelles, including anterograde and retrograde regulation, have been actively investigated in the last two decades. Norflurazon (NF) and lincomycin (Lin) are two commonly used inhibitors to study chloroplast-to-nucleus retrograde signaling in plants. We used NF and Lin to block the development and functions of chloroplasts and examined their effects on mitochondrial gene expression, RNA editing and splicing. The editing of most mitochondrial transcripts was not affected, but the editing extents of nad4-107, nad6-103, and ccmFc-1172 decreased slightly in NF- and Lin-treated seedlings. While the splicing of mitochondrial transcripts was not significantly affected, steady-state mRNA levels of several mitochondrial genes increased significantly in NF- and Lin-treated seedlings. Moreover, Lin seemed to have more profound effects than NF on the expression of mitochondrial genes, indicating that signals derived from these two inhibitors might be distinct. NF and Lin also significantly induced the expression of nuclear genes encoding subunits of mitochondrial electron transport chain complexes. Thus, dysfunctional chloroplasts may coordinately up-regulate the expression of nuclear and mitochondrial genes encoding subunits of respiratory complexes.

Gene expression profiling of rice seedlings in response to glutamine treatment.

Glutamine, the most abundant free amino acid in humans (Curi et al., 2007 [1]), has many functions. In addition to protein, amino acid, and nucleic acid biosynthesis, glutamine also regulates the expression of genes related to metabolism, cell defense, and signal transduction in humans (Curi et al., 2007 [1]; Brasse-Lagnel et al., 2009 [2]). Glutamine is also one of the major forms of nitrogen in rice (Fukumorita and Chino, 1982 [3]). In addition to metabolic and nutritional effects, glutamine may function as a signaling molecule to regulate gene expression in plants. To this end, we used microarray analysis to identify genes that are rapidly induced by 2.5 mM glutamine in rice roots. The results revealed that glutamine induced the expression of at least 35 genes involved in metabolism, transport, signal transduction, and stress responses within 30 min (Kan et al., 2015 [4]). Here, we provide the details of the experimental procedure associated with our microarray data deposited in NCBI's Gene Expression Omnibus (GEO ID: GSE56770).

Glutamine rapidly induces the expression of key transcription factor genes involved in nitrogen and stress responses in rice roots.

BACKGROUND: Glutamine is a major amino donor for the synthesis of amino acids, nucleotides, and other nitrogen-containing compounds in all organisms. In addition to its role in nutrition and metabolism, glutamine can also function as a signaling molecule in bacteria, yeast, and humans. By contrast, the functions of glutamine in nutrition and as a signaling molecule remain unclear in plants. RESULTS: We demonstrated that glutamine could effectively support the growth of rice seedlings. In glutamine-treated rice roots, the glutamine contents increased dramatically, whereas levels of glutamate remained relatively constant. Transcriptome analysis of rice roots revealed that glutamine induced the expression of at least 35 genes involved in metabolism, transport, signal transduction, and stress responses within 30 min. Interestingly, 10 of the 35 early glutamine responsive genes encode putative transcription factors, including two LBD37-like genes that are involved in the regulation of nitrogen metabolism. Glutamine also rapidly induced the expression of the DREB1A, IRO2, and NAC5 transcription factor genes, which are involved in the regulation of stress responses. CONCLUSIONS: In addition to its role as a metabolic fuel, glutamine may also function as a signaling molecule to regulate gene expression in plants. The rapid induction of transcription factor genes suggests that glutamine may efficiently amplify its signal and interact with the other signal transduction pathways to regulate plant growth and stress responses. Thus, glutamine is a functional amino acid that plays important roles in plant nutrition and signal transduction.