Vegetative Growth In Arabidopsis Research Articles

Nitrate-responsive NIN-like protein transcription factors perform unique and redundant roles in Arabidopsis.

Upon sensing nitrate, NODULE INCEPTION (NIN)-like protein (NLP) transcription factors alter gene expression to promote nitrate uptake and utilization. Of the nine NLPs in Arabidopsis, the physiological roles of only three NLPs (NLP6-NLP8) have been characterized to date. To evaluate the unique and redundant roles of Arabidopsis NLPs, we assessed the phenotypes of single and higher order nlp mutants. Unlike other nlp single mutants, nlp2 and nlp7 single mutants showed a reduction in shoot fresh weight when grown in the presence of nitrate as the sole nitrogen source, indicating that NLP2, like NLP7, plays a major role in vegetative growth. Interestingly, the growth defect of nlp7 recovered upon the supply of ammonium or glutamine, whereas that of nlp2 did not. Furthermore, complementation assays using chimeric constructs revealed that the coding sequence, but not the promoter region, of NLP genes was responsible for the differences between nlp2 and nlp7 single mutant phenotypes, suggesting differences in protein function. Importantly, nitrate utilization was almost completely abolished in the nlp septuple mutant (nlp2 nlp4 nlp5 nlp6 nlp7 nlp8 nlp9), suggesting that NLPs other than NLP2 and NLP7 also assist in the regulation of nitrate-inducible gene expression and nitrate-dependent promotion of vegetative growth in Arabidopsis.

The Arabidopsis SAL1-PAP Pathway: A Case Study for Integrating Chloroplast Retrograde, Light and Hormonal Signaling in Modulating Plant Growth and Development?

Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3′-phosphoadenosine-5′-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.

Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development.

The LAFL (i.e. LEC1, ABI3, FUS3, and LEC2) master transcriptional regulators interact to form different complexes that induce embryo development and maturation, and inhibit seed germination and vegetative growth in Arabidopsis. Orthologous genes involved in similar regulatory processes have been described in various angiosperms including important crop species. Consistent with a prominent role of the LAFL regulators in triggering and maintaining embryonic cell fate, their expression appears finely tuned in different tissues during seed development and tightly repressed in vegetative tissues by a surprisingly high number of genetic and epigenetic factors. Partial functional redundancies and intricate feedback regulations of the LAFL have hampered the elucidation of the underpinning molecular mechanisms. Nevertheless, genetic, genomic, cellular, molecular, and biochemical analyses implemented during the last years have greatly improved our knowledge of the LALF network. Here we summarize and discuss recent progress, together with current issues required to gain a comprehensive insight into the network, including the emerging function of LEC1 and possibly LEC2 as pioneer transcription factors.

HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition from Seed Maturation to Vegetative Growth in Arabidopsis.

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

Clathrin Heavy Chain Subunits Coordinate Endo- and Exocytic Traffic and Affect Stomatal Movement.

The current model for vesicular traffic to and from the plasma membrane is accepted, but the molecular requirements for this coordination are not well defined. We have identified the hot ABA-deficiency suppressor1 mutant, which has a stomatal function defect, as a clathrin heavy chain1 (CHC1) mutant allele and show that it has a decreased rate of endocytosis and growth defects that are shared with other chc1 mutant alleles. We used chc1 alleles and the related chc2 mutant as tools to investigate the effects that clathrin defects have on secretion pathways and plant growth. We show that secretion and endocytosis at the plasma membrane are sensitive to CHC1 and CHC2 function in seedling roots and that chc mutants have physiological defects in stomatal function and plant growth that have not been described previously. These findings suggest that clathrin supports specific functions in multiple cell types. Stomata movement and gas exchange are altered in chc mutants, indicating that clathrin is important for stomatal regulation. The aberrant function of chc mutant stomata is consistent with the growth phenotypes observed under different water and light conditions, which also are similar to those of the secretory SNARE mutant, syp121 The syp121 and chc mutants have impaired endocytosis and exocytosis compared with the wild type, indicating a link between SYP121-dependent secretion and clathrin-dependent endocytosis at the plasma membrane. Our findings provide evidence that clathrin and SYP121 functions are important for the coordination of endocytosis and exocytosis and have an impact on stomatal function, gas exchange, and vegetative growth in Arabidopsis (Arabidopsis thaliana).

Contribution of PPi-Hydrolyzing Function of Vacuolar H(+)-Pyrophosphatase in Vegetative Growth of Arabidopsis: Evidenced by Expression of Uncoupling Mutated Enzymes.

The vacuolar-type H+-pyrophosphatase (H+-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the tonoplast. Overexpression of H+-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H+-PPase in Arabidopsis thaliana have post-germinative developmental defects. Here, to further clarify the physiological significance of this important enzyme, we newly generated three varieties of H+-PPase overexpressing lines with different levels of activity that we analyzed together with the loss-of-function mutant fugu5-3. The H+-PPase overexpressors exhibited enhanced activity of H+-PPase during vegetative growth, but no change in the activity of vacuolar H+-ATPase. Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively. Consistently, the overexpressors had larger rosette leaves and nearly 30% more cells in leaves than the wild type. When uncoupling mutated variants of H+-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H+-PPase was introduced. Taken together, our findings clearly demonstrate that additional expression of H+-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis.

In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. In this study, we identified that the Arabidopsis Transmembrane Protein 18 (AtTMEM18) gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins (TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen‐rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18‐GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility. Furthermore, expression of the rice TMEM18‐homologous protein (OsTMEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.

EXTENSIN18 is required for full male fertility as well as normal vegetative growth in Arabidopsis.

EXTENSINS (EXTs) are a 65-member subfamily of hydroxyproline-rich glycoproteins (HRGPs) of which 20 putatively form crosslinking networks in the cell wall. These 20 classical EXTs are involved at the start of new wall assembly as evidenced by a requirement for EXT3 during cytokinesis, and the ability of some EXTs to polymerize in vitro into dendritic patterns. EXT3 was previously shown to form pulcherosine (three Tyrosines) cross-links. Little direct data exists on the other 19 classical EXTs. Here, we describe the phenotypes of ext18 mutants and rescued progeny as well as associated expression profiles of all 20 classical EXT genes. We found that EXT18 is required for full male fertility, as well as for normal vegetative growth. EXT18 has potential to form crosslinking networks via di-iso-di-tyrosine (four Tyrosines) covalent bonds, and not via pulcherosine due to deficit of lone Tyrosines. This together with ext18 defective pollen grains and pollen tubes, and reduced plant size, suggests that EXT18-type EXTs are important contributors to wall integrity, in pollen and other rapidly extending walls. The data also show that a knockout of EXT18 had a pleiotropic affect on the expression of several EXTs, as did the reintroduction of the native EXT18 gene, thus supporting the thesis that transcription of groups of EXTs are co-regulated and work in different combinations to make distinctive inputs into wall assembly of different cell types. These insights contribute to basic knowledge of cell wall self-assembly in different cell types, and potentially enable biotechnological advances in biomass increase and plant fertility control.

Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis.

BackgroundEco1/Ctf7 is essential for the establishment of sister chromatid cohesion during S phase of the cell cycle. Inactivation of Ctf7/Eco1 leads to a lethal phenotype in most organisms. Altering Eco1/Ctf7 levels or point mutations in the gene can lead to alterations in nuclear division as well as a wide range of developmental defects. Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth.ResultsTo further investigate the function(s) of AtCTF7, a tagged version of AtCTF7 and several AtCTF7 deletion constructs were created and transformed into wild type or ctf7+/–plants. Transgenic plants expressing 35S:NTAP:AtCTF7∆299–345 (AtCTF7∆B) displayed a wide range of phenotypic alterations in reproduction and vegetative growth. Male meiocytes exhibited chromosome fragmentation and uneven chromosome segregation. Mutant ovules contained abnormal megasporocyte-like cells during pre-meiosis, megaspores experienced elongated meiosis and megagametogenesis, and defective megaspores/embryo sacs were produced at various stages. The transgenic plants also exhibited a broad range of vegetative defects, including meristem disruption and dwarfism that were inherited in a non-Mendelian fashion. Transcripts for epigenetically regulated transposable elements (TEs) were elevated in transgenic plants. Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B.ConclusionsHigh level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0452-2) contains supplementary material, which is available to authorized users.

Erratum to: Poplar GATA transcription factor PdGNC is capable of regulating chloroplast ultrastructure, photosynthesis, and vegetative growth in Arabidopsis under varying nitrogen levels

Erratum to: Poplar GATA transcription factor PdGNC is capable of regulating chloroplast ultrastructure, photosynthesis, and vegetative growth in Arabidopsis under varying nitrogen levels

Poplar GATA transcription factor PdGNC is capable of regulating chloroplast ultrastructure, photosynthesis, and vegetative growth in Arabidopsis under varying nitrogen levels

Photosynthesis and nitrogen availability are crucial to the accumulation of biomass. The GATA transcription factor family plays important roles in chloroplast development and nitrogen metabolism. Here, we cloned, for the first time, the GATA transcription factor PdGNC from the fast-growing poplar clone NE-19. The overexpression results from Arabidopsis under high nitrate, sufficient nitrate, and low nitrate (LN) support that PdGNC increased the chloroplast number and size per cell in leaf and stem, improved the chlorophyll level by 26.12 % and exhibited the highest starch content in LN. Overexpression of PdGNC also had pronounced effects on chloroplast ultrastructure by increasing the number of grana and thylakoids. The photosynthetic rate in transgenic LN lines was 42.17 % higher than in the wild type through modification of the chlorophyll fluorescence parameters Fv/F0, Fv/Fm, qP, NPQ, and ΦPSII. Morphologically, PdGNC promoted longer primary roots and larger leaf areas, and exhibited a higher relative growth rate in LN. Altogether, PdGNC improved photosynthetic capacity and plant growth under low nitrate levels; thus, it could potentially be used in transgenic breeding to improve nitrate utilization and plant growth rates under limited nitrogen conditions.

The Effect of a Genetically Reduced Plasma Membrane Protonmotive Force on Vegetative Growth of Arabidopsis

The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H(+)-ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.

Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2′3′-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.

BROTHER OF FT AND TFL1 (BFT), a member of the FT/TFL1 family, shows distinct pattern of expression during the vegetative growth of Arabidopsis

Transition to the flowering stage is precisely controlled by a few classes of regulatory molecules. BROTHER OF FT AND TFL1 (BFT) is a member of FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, an important class of flower development regulators with unidentified biochemical function. BFT has a TFL1-like activity and plays a role in axillary inflorescence development. To elucidate the expression pattern of BFT, we analyzed the subcellular localization and conditional expression of BFT in this study. We generated 35S::BFT:GFP plants to investigate the subcellular localization of BFT protein. 35S::BFT:GFP plants showed late flowering, similarly as did 35S::BFT plants. BFT:GFP fusion protein was localized in the nucleus and the plasma membrane, which was different from the localization pattern of FT and TFL1. BFT expression was induced by abiotic stress conditions. ABA, drought, and osmotic stress treatments induced BFT expression, whereas cold, salt, and heat stress conditions did not, suggesting that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions. The induction pattern of BFT was different from those of other FT/TFL1 family genes. Our studies indicated that BFT showed a distinct expression pattern from its homologous genes during the vegetative growth in Arabidopsis.

Protist Homologs of the Meiotic Spo11 Gene and Topoisomerase VI reveal an Evolutionary History of Gene Duplication and Lineage-Specific Loss

Spo11 is a meiotic protein of fundamental importance as it is a conserved meiosis-specific transesterase required for meiotic recombination initiation in fungi, animals, and plants. Spo11 is homologous to the archaebacterial topoisomerase VIA (Top6A) gene, and its homologs are broadly distributed among eukaryotes, with some eukaryotes having more than one homolog. However, the evolutionary relationships among these genes are unclear, with some debate as to whether eukaryotic homologs originated by lateral gene transfer. We have identified and characterized protist Spo11 homologs by degenerate polymerase chain reaction (PCR) and sequencing and by analyses of sequences from public databases. Our phylogenetic analyses show that Spo11 homologs evolved by two ancient eukaryotic gene duplication events prior to the last common ancestor of extant eukaryotes, resulting in three eukaryotic paralogs: Spo11-1, Spo11-2, and Spo11-3. Spo11-1 orthologs encode meiosis-specific proteins and are distributed broadly among eukaryotic lineages, though Spo11-1 is absent from some protists. This absence coincides with the presence of Spo11-2 orthologs, which are meiosis-specific in Arabidopsis and are found in plants, red algae, and some protists but absent in animals and fungi. Spo11-3 encodes a Top6A subunit that interacts with topoisomerase VIB (Top6B) subunits, which together play a role in vegetative growth in Arabidopsis. We identified Spo11-3 (Top6A) and Top6B homologs in plants, red algae, and a few protists, establishing a broader distribution of these genes among eukaryotes, indicating their likely vertical descent followed by lineage-specific loss.

Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana

Three metabolites of indole-3-acetic acid (IAA), N-(6-hydroxyindol-3-ylacetyl)-phenylalanine (6-OH-IAA-Phe), N-(6-hydroxyindol-3-ylacetyl)-valine (6-OH-IAA-Val), and 1- O-(2-oxoindol-3-ylacetyl)-β- d-glucopyranose (OxIAA-Glc), were found by a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based search for oxidative IAA metabolites during the vegetative growth of Arabidopsis. Their structures were confirmed by making a comparison of chromatographic characteristics and mass spectra between naturally occurring compounds and synthetic standards. An incorporation study using deuterium-labeled compounds showed that 6-OH-IAA-Phe and 6-OH-IAA-Val were biosynthesized from IAA-Phe and IAA-Val, respectively, which strongly suggested the formation of these amino acid conjugates of IAA in plants. Both 6-OH-IAA-Phe and 6-OH-IAA-Val were inactive as auxins, as indicated by no significant root growth inhibition in Arabidopsis. Quantitative analysis demonstrated that OxIAA-Glc was present in the largest amount among the metabolites of IAA in Arabidopsis, suggesting that the conversion into OxIAA-Glc represents the main metabolic process regarding IAA in Arabidopsis.

A new member of the LIR gene family from perennial ryegrass is cold-responsive, and promotes vegetative growth in Arabidopsis

A cold-regulated gene Lolium perenne LIR1 ( LpLIR1) was isolated from perennial ryegrass using a subtractive approach. The gene has strong homology to the Light Induced Rice1 ( LIR1) gene and is regulated at the transcriptional level by cold, and by a diurnal rhythm. Expression of LpLIR1 in perennial ryegrass was upregulated by vernalization but did not follow a standard vernalization-responsive expression pattern. LpLIR1 expression was restricted to vegetative tissues and absent in apices during floral induction and in flowers. LpLIR1 mRNA levels displayed diurnal fluctuations, which peaked before dusk and declined during the night. Heterologous expression of LpLIR1 in Arabidopsis led to a significant increase in leaf formation under short days (SD) conditions but only when plants had received a preceding vernalization treatment. Furthermore, dissection of plant development under SD revealed a minor but significant delay of flowering in the transgenic lines compared to wildtype plants.

Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

A general gas chromatography/mass spectrometry (MS)-based screen was performed to identify catabolites and conjugates of indole-3-acetic acid (IAA) during vegetative growth of Arabidopsis. This experiment revealed the existence of two new conjugates:N-(indole-3-acetyl)-alfa-alanine (IA-Ala) andN-(indole-3-acetyl)-alfa-leucine (IA-Leu). A method for quantitative analysis of IAA metabolites in plant extracts by liquid chromatography-electrospray tandem MS has been developed. The accuracy and precision of the new method are better than 10% for standards close to the detection limit, and are between 6% and 16% for the entire protocol applied to plant extracts. The low detection limits, 0.02 to 0.1 pmol for the different metabolites, made it possible to use as little as 50 to 100 mg of tissue for quantitative analysis. The analysis was performed on different tissues of an Arabidopsis plant at two stages of development, using heavy labeled internal standards of the catabolite 2-oxoindole-3-acetic acid as well as IAA conjugated to amino acids: aspartate, glutamate, Ala, and Leu. Expanding leaves and roots that generally contain high amounts of the free hormone also contained the highest levels of IA-aspartate, IA-glutamate, and 2-oxoindole-3-acetic acid, supporting their role as irreversible catabolic products. The levels of IA-Leu and IA-Ala did not follow the general distribution of IAA. Interestingly, the level of IA-Leu was highest in roots and IA-Ala in the aerial tissues.

Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

A general gas chromatography/mass spectrometry (MS)-based screen was performed to identify catabolites and conjugates of indole-3-acetic acid (IAA) during vegetative growth of Arabidopsis. This experiment revealed the existence of two new conjugates: N-(indole-3-acetyl)-alfa-alanine (IA-Ala) and N-(indole-3-acetyl)-alfa-leucine (IA-Leu). A method for quantitative analysis of IAA metabolites in plant extracts by liquid chromatography-electrospray tandem MS has been developed. The accuracy and precision of the new method are better than 10% for standards close to the detection limit, and are between 6% and 16% for the entire protocol applied to plant extracts. The low detection limits, 0.02 to 0.1 pmol for the different metabolites, made it possible to use as little as 50 to 100 mg of tissue for quantitative analysis. The analysis was performed on different tissues of an Arabidopsis plant at two stages of development, using heavy labeled internal standards of the catabolite 2-oxoindole-3-acetic acid as well as IAA conjugated to amino acids: aspartate, glutamate, Ala, and Leu. Expanding leaves and roots that generally contain high amounts of the free hormone also contained the highest levels of IA-aspartate, IA-glutamate, and 2-oxoindole-3-acetic acid, supporting their role as irreversible catabolic products. The levels of IA-Leu and IA-Ala did not follow the general distribution of IAA. Interestingly, the level of IA-Leu was highest in roots and IA-Ala in the aerial tissues.