Zhenyu Cheng

Promotion of Plant Growth by Bacterial ACC Deaminase

To date, there has been only limited commercial use of plant growth-promoting bacteria in agriculture, horticulture, and silviculture. However, with recent progress toward understanding the mechanisms that these organisms utilize to facilitate plant growth, the use of plant growth-promoting bacteria is expected to continue to increase worldwide. One of the key mechanisms employed by plant growth-promoting bacteria to facilitate plant growth is the lowering of plant ethylene levels by the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase. This article reviews the published work on this enzyme, with an emphasis on its biochemistry, protein structure, genes, and regulation. In addition, this article provides some initial insights into the changes in both plants and ACC deaminase-containing plant growth-promoting bacteria as a consequence of plant-microbe interactions. Finally, a brief discussion of how bacterial ACC deaminase and indoleacetic acid (IAA) together modulate plant growth and development is included.

Promotion of plant growth by ACC deaminase-producing soil bacteria

Plant growth-promoting bacteria that contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase facilitate plant growth and development by decreasing plant ethylene levels, especially following a variety of environmental stresses. In this review, the physiological basis for this growth-promotion effect is examined in some detail. In addition, models are presented that endeavour to explain (i) the seemingly paradoxical effects of ethylene on a plant’s response to stress, (ii) how the expression of this enzyme is transcriptionally regulated in many bacterial strains and (iii) how ACC deaminase-containing plant growth-promoting bacteria alter plant gene expression and positively modulate plant growth.

The presence of a 1-aminocyclopropane-1-carboxylate (ACC) deaminase deletion mutation alters the physiology of the endophytic plant growth-promoting bacterium Burkholderia phytofirmans PsJN

The structural gene for 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) from the endophytic plant growth-promoting bacterium Burkholderia phytofirmans PsJN was isolated and used to construct a mutant strain B. phytofirmans YS2 (B. phytofirmans PsJN/Delta acdS), in which an internal segment of the acdS gene was deleted. The mutant YS2 lost ACC deaminase activity as well as the ability to promote the elongation of the roots of canola seedlings. Concomitant with the creation of this deletion mutant, a number of physiological changes were observed in the bacterium, including an increase in indole acetic acid synthesis, a decrease in the production of siderophores and an increase in the cellular level of the stationary-phase sigma factor, RpoS. Introduction of the wild-type acdS gene into the mutant YS2 to construct strain B. phytofirmans YS3 (B. phytofirmans YS2/pRK-AcdS) restored both ACC deaminase activity and plant growth-promotion activity in strain YS3. However, the complemented mutant still showed the above-mentioned physiological changes.

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup.

Identification of Bacterial Proteins Mediating the Interactions Between Pseudomonas putida UW4 and Brassica napus (Canola)

The influence of canola root exudates on the proteome of Pseudomonas putida UW4 and the mutant strain P. putida UW4/AcdS(-), which lacks a functional 1-aminocyclopropane-1-carboxylate deaminase gene, was examined using two-dimensional difference in-gel electrophoresis. Seventy-two proteins with significantly altered expression levels in the presence of canola root exudates were identified by mass spectrometry. Many of these proteins are involved in nutrient transport and utilization, cell envelope synthesis, and transcriptional or translational regulation and, hence, may play important roles in plant-bacterial interactions. Four proteins showing large changes in expression in response to canola root exudates in both the wild-type and mutant strains of P. putida UW4 (i.e., outer membrane protein F, peptide deformylase, transcription regulator Fis family protein, and a previously uncharacterized protein) were both overexpressed and disrupted in P. putida UW4 in an effort to better understand their functions. Functional studies of these modified strains revealed significantly enhanced or inhibited plant-growth-promoting abilities compared with the wild-type P. putida UW4, in agreement with the suggested involvement of three of these four proteins in plant-bacterial interactions. The work reported here suggests strategies to both identify potential antibacterial agents and develop bacterial strains that might be useful adjuncts to agriculture. This approach may be an effective means of identifying key proteins mediating the interactions of bacteria with their rhizosphere environment.

Proteomic analysis of the response of the plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress

BackgroundPlant growth-promoting bacteria can alleviate the inhibitory effects of various heavy metals on plant growth, via decreasing levels of stress-induced ethylene. However, little has been done to detect any mechanisms specific for heavy metal resistance of this kind of bacteria. Here, we investigate the response of the wild-type plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress using proteomic approaches. The mutant strain P. putida UW4/AcdS-, lacking a functional 1-aminocyclopropane-1-carboxylic acid deaminase gene, was also assessed for its response to nickel stress.ResultsTwo dimensional difference in-gel electrophoresis (DIGE) was used to detect significantly up- or down- regulated proteins (p < 0.05, | ratio | > 1.5) in P. putida in response to the presence of 2 mM Ni. Out of a total number of 1,702 proteins detected on the analytical gels for P. putida UW4, the expression levels of 82 (4.82%) proteins increased significantly while the expression of 81 (4.76%) proteins decreased significantly. Of 1,575 proteins detected on the analytical gels for P. putida UW4/AcdS-, the expression levels of 74 (4.70%) proteins increased and 51 (3.24%) proteins decreased significantly. Thirty-five proteins whose expression was altered were successfully identified by mass spectrometry and sequence comparisons with related species. Nineteen of the identified proteins were detected as differentially expressed in both wild-type and mutant expression profiles.ConclusionFunctional assessment of proteins with significantly altered expression levels revealed several mechanisms thought to be involved in bacterial heavy metal detoxification, including general stress adaptation, anti-oxidative stress and heavy metal efflux proteins. This information may contribute to the development of plant growth-promoting bacteria mediated phytoremediation processes.

Identification of plant growth-promoting bacteria-responsive proteins in cucumber roots under hypoxic stress using a proteomic approach.

UNLABELLED Plant growth-promoting bacteria (PGPB) can both facilitate plant growth and improve plant resistance to a variety of environmental stresses. In order to investigate the mechanisms that PGPB use to protect plants under hypoxic conditions, the protein profiles of stressed and non-stressed cucumber roots, either treated or not treated with PGPB, were examined. Two dimensional difference in-gel electrophoresis (DIGE) was used to detect significantly up- or down-regulated proteins (p<0.05, |ratio|>1.5) in cucumber roots in response to hypoxia. There were 1980, 1893 and 1735 protein spots detected from cucumber roots in the absence of stress in the presence of the PGPB Pseudomonas putida UW4, following hypoxic stress, and following hypoxic stress in the presence of P. putida UW4, respectively. The numbers of significantly changed protein spots were 0, 106, and 147 in these three treatments respectively. Proteins were identified by LTQ-MS/MS and categorized into classes corresponding to transcription, protein synthesis, signal transduction, carbohydrate and nitrogen metabolism, defense stress, antioxidant, binding and others. The functions of the proteins whose expression changed significantly were analyzed in detail, contributing to a more thorough understanding of how PGPB mediate the stress response in plants. BIOLOGICAL SIGNIFICANCE To our knowledge, only a limited number of papers have addressed cucumber proteomics, this study is the first report to describe the effect of plant growth-promoting bacteria (P. putida UW4) on cucumber plants under hypoxic stress using a proteomic approach. Thus, this work provides new insights to understand the cross-reactivity between P. putida UW4 and cucumber plant. A model of cucumber roots in response to P. putida UW4 and hypoxia was proposed: P. putida UW4 and hypoxic stress caused changes of gene expression in cucumber roots, then transcription was stimulated, the proteins involved in carbohydrate metabolism, nitrogen metabolism, defense stress, antioxidant, binding and others were induced, these proteins might work cooperatively to release hypoxic stress and promote cucumber growth. These results describe a dynamic protein network to explain the promotion mechanism of P. putida UW4, and also provide a solid basis for further functional research of single nodes of this network.

Proteome reference map for the plant growth-promoting bacterium Pseudomonas putida UW4.

A proteome reference map containing 326 2‐D gel spots representing 275 different proteins was constructed for the plant growth‐promoting bacterium Pseudomonas putida UW4. Protein identifications were obtained using Q‐TOF MS/MS spectra matching to homologous proteins from other Pseudomonas strains and confirmed by PMF analysis. This data set is accessible at http://world‐2dpage.expasy.org/repository/ and will aid in further characterization of Pseudomonas strains and interactions of plant growth‐promoting bacterium with the plant rhizosphere environment.

Investigating the Role of Protein UnkG from the Pseudomonas putida UW4 in the Ability of the Bacterium to Facilitate Plant Growth

A previous study showed that overexpressing protein UnkG decreased the ability of the plant growth-promoting bacterium Pseudomonas putida UW4 to facilitate plant growth and an unkG knockout mutant of P. putida UW4 displayed increased plant growth promotion. When activities of wild-type and the UnkG overexpressing strain, including growth rates, carbon utilization, cell size, 3-indoleacetic acid production, and 1-aminocyclopropane-1-carboxylate deaminase activity, were measured, there were no apparent differences between the strains. Monitoring proteome-level changes to the wild-type and overexpressing transformant by means of two-dimensional difference in-gel electrophoresis followed by mass spectrometry identification of the altered proteins, 1839 protein spots were detected and 16 of the 84 protein spots with changed expression levels were identified. Proteins with increased expression included arginine deiminase, dihydrodipicolinate synthase, azurin, flavoprotein (α-subunit), ferredoxin-NADP reductase, ATP-dependent Hs1 protease (ATP-binding subunit), UDP-N-acetyl muramate-l-alanine ligase, biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase, and Fis two-component transcriptional regulator. Proteins with decreased expression included glutaminase-asparaginase, arginine/ornithine ABC transporter, cell division protein FtsZ and glutamyl-tRNA synthetase. The functions of three of the 16 proteins could not be identified. The results are consistent with UnkG being detrimental to plant growth because it acts as a regulatory protein that negatively affects several key cellular functions related to the energy balance of the bacterium.