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Plant roots are colonized by an immense number of microbes, referred to as the root microbiome. Selected strains of beneficial soil-borne bacteria can protect against abiotic stress and prime the plant immune system against a broad range of pathogens. Pseudomonas spp. rhizobacteria represent one of the most abundant genera of the root microbiome. Here, by employing a germ-free experimental system, we demonstrate the ability of selected Pseudomonas spp. strains to promote plant growth and drive developmental plasticity in the roots of Arabidopsis (Arabidopsis thaliana) by inhibiting primary root elongation and promoting lateral root and root hair formation. By studying cell type-specific developmental markers and employing genetic and pharmacological approaches, we demonstrate the crucial role of auxin signaling and transport in rhizobacteria-stimulated changes in the root system architecture of Arabidopsis. We further show that Pseudomonas spp.-elicited alterations in root morphology and rhizobacteria-mediated systemic immunity are mediated by distinct signaling pathways. This study sheds new light on the ability of soil-borne beneficial bacteria to interfere with postembryonic root developmental programs.

To develop a growing root, cell division in the root meristem has to be properly regulated in order to generate or propagate new cells. How cell division is regulated in the root meristem remains largely unknown. Here, we report the identification and characterization of the Arabidopsis (Arabidopsis thaliana) RETARDED ROOT GROWTH (RRG) gene that plays a role in the regulation of root meristem cell division. In the root, RRG is predominantly expressed in the root meristem. Disruption of RRG function reduced numbers of dividing cells, the rate of cell production, and endoreduplication, and thus affected meristem size and root growth. Quantitative reverse transcription-polymerase chain reaction and marker-assisted analyses revealed that expression levels of several cell cycle genes were decreased in the mutant roots, indicating a defect in cell cycle progression. Mutations in RRG, however, did not affect the expression of key root-patterning genes and an auxin-responsive marker, suggesting that RRG is not essential for root patterning and auxin signaling. RRG is a mitochondria-localized protein conserved in plants and shares a DUF155 domain with proteins related to cell division in yeast, and rrg mutants displayed extensive vacuolization in mitochondria. We propose that Arabidopsis RRG is a conserved mitochondrial protein required for cell division in the root meristem.

Jasmonic acid (JA) and ethylene (ET) are known to play important roles in mediating plant defense against herbivores, but how they affect development in herbivore-attacked plants is unknown. We used JA-deficient (silenced in LIPOXYGENASE3 [asLOX3]) and ET-insensitive (expressing a mutated dominant negative form of ETHYLENE RESPONSE1 [mETR1]) Nicotiana attenuata plants, and their genetic cross (mETR1asLOX3), to examine growth and development of these plants under simulated herbivory conditions. At the whole plant level, both hormones suppressed leaf expansion after the plants had been wounded and the wounds had been immediately treated with Manduca sexta oral secretions (OS). In addition, ectopic cell expansion was observed around both water- and OS-treated wounds in mETR1asLOX3 leaves but not in mETR1, asLOX3, or wild-type leaves. Pretreating asLOX3 leaves with the ET receptor antagonist 1-methylcyclopropane resulted in local cell expansion that closely mimicked the mETR1asLOX3 phenotype. We found higher auxin (indole-3-acetic acid) levels in the elicited leaves of mETR1asLOX3 plants, a trait that is putatively associated with enhanced cell expansion and leaf growth in this genotype. Transcript profiling of OS-elicited mETR1asLOX3 leaves revealed a preferential accumulation of transcripts known to function in cell wall remodeling, suggesting that both JA and ET act as negative regulators of these genes. We propose that in N. attenuata, JA-ET cross talk restrains local cell expansion and growth after herbivore attack, allowing more resources to be allocated to induced defenses against herbivores.

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.

Amide-linked indole-3-acetic acid (IAA) conjugates constitute approximately 90% of the IAA pool in the dicot Arabidopsis, whereas ester-linked conjugates and free IAA account for approximately 10% and 1%, respectively when whole seedlings are measured. We show here that IAA-aspartate Asp, IAA-glutamate (Glu), and IAA-glucose (Glc) are present at low levels in Arabidopsis. Nine-day-old wild-type Arabidopsis seedlings yielded 17.4 +/- 4.6 ng g(-1) fresh weight IAA-Asp and 3.5 +/- 1.6 ng g(-1) fresh weight IAA-Glu, and IAA-Glc was present at 7 to 17 ng g(-1) fresh weight in 12-d-old wild-type seedlings. Total IAA content in 9-d-old Arabidopsis seedlings was 1, 200 +/- 178 ng g(-1) fresh weight, so these three IAA conjugates together made up only 3% of the conjugate pool throughout the whole plant. We detected less than wild-type levels of IAA-Asp and IAA-Glu (7.8 +/- 0.4 ng g(-1) fresh weight and 1.8 +/- 0.3 ng g(-1) fresh weight, respectively) in an Arabidopsis mutant that accumulates conjugated IAA. Our results are consistent with IAA-Asp, IAA-Glu, and IAA-Glc being either minor, transient, or specifically localized IAA metabolites under normal growth conditions and bring into question the physiological relevance of IAA-Asp accumulation in response to high concentrations of exogenous IAA.

The genetic advantages to the use of Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and indentification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAA]) was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of 13C1-labeled IAN and its utility in the gas chromatography-mass spectrometry quantification of endogenous IAN levels. We also quantified the nonenzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. 13C1-Labeled IAN was used to assess the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.

Measurements were made of the fresh weight, dry weight, dry weight-fresh weight ratio, free and conjugated indole-3-acetic acid, and free and conjugated abscisic acid in seedlings of Zea mays grown in darkness in microgravity and on earth. Imbibition of the dry kernels was 17 h prior to launch. Growth was for 5 d at ambient orbiter temperature and at a chronic accelerational force of the order of 3 x 10(-5) times earth gravity. Weights and hormone content of the microgravity seedlings were, with minor exceptions, not statistically different from seedlings grown in normal gravity. The tissues of the shuttle-grown plants appeared normal and the seedlings differed only in the lack of orientation of roots and shoots. These findings, based upon 5 d of growth in microgravity, cannot be extrapolated to growth in microgravity for weeks, months, and years, as might occur on a space station. Nonetheless, it is encouraging, for prospects of bioregeneration of the atmosphere and food production in a space station, that no pronounced differences in the parameters measured were apparent during the 5 d of plant seedling growth in microgravity.

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

Acidic indole auxins have been extracted from N. glauca, N. langsdorffii and their 2 tumor-prone 4n- and 2n-hybrids. After purification of the extracts and thin-layer chromatography, acidic indoles were subjected to esterification and gas chromatography. The esters of 4 indole acids were detected and determined: indole-3-acetic acid, indole-3-carboxylic acid, indole-3-propionic acid and indole-3-butyric acid. The indolic nature of fractionated samples was confirmed by spectrophotofluorometry and the physiological significance of the indole esters proven in a biotest. A substantial increase in extractable indole-3-butyric acid in the tumor-prone hybrids suggests an additional pathway of auxin synthesis in these tissues.