Peter Weisbeek

An Auxin-Dependent Distal Organizer of Pattern and Polarity in the Arabidopsis Root

Root formation in plants involves the continuous interpretation of positional cues. Physiological studies have linked root formation to auxins. An auxin response element displays a maximum in the Arabidopsis root and we investigate its developmental significance. Auxin response mutants reduce the maximum or its perception, and interfere with distal root patterning. Polar auxin transport mutants affect its localization and distal pattern. Polar auxin transport inhibitors cause dramatic relocalization of the maximum, and associated changes in pattern and polarity. Auxin application and laser ablations correlate root pattern with a maximum adjacent to the vascular bundle. Our data indicate that an auxin maximum at a vascular boundary establishes a distal organizer in the root.

A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r–mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid–nonaccumulating NahG plants. Moreover, methyl jasmonate–induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate–induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

Short-range control of cell differentiation in the Arabidopsis root meristem

Meristems are distinctive regions of plants that have capacity for continuous growth. Their developmental activity generates the majority of plant organs. It is currently unknown how cell division and cell differentiation are orchestrated in meristems, although genetic studies have demonstrated the relevance of a proper balance between the two processes. Root meristems contain a distinct central region of mitotically inactive cells, the quiescent centre, the function of which has remained elusive until now. Here we present laser ablation and genetic data that show that in Arabidopsis thaliana the quiescent centre inhibits differentiation of surrounding cells. Differentiation regulation occurs within the range of a single cell, in a manner strikingly similar to examples in animal development, such as during delamination of Drosophila neuroblasts. Our data indicate that pattern formation in the root meristem is controlled by a balance between short-range signals inhibiting differentiation and signals that reinforce cell fate decisions.

Improved Performance of Transgenic Fructan-Accumulating Tobacco under Drought Stress.

Fructans are polyfructose molecules produced by approximately 15% of the flowering plant species. It is possible that, in addition to being a storage carbohydrate, fructans have other physiological roles. Owing to their solubility they may help plants survive periods of osmotic stress induced by drought or cold. To investigate the possible functional significance of fructans, use was made of transgenic tobacco (Nicotiana tabacum) plants that accumulate bacterial fructans and hence possess an extra sink for carbohydrate. Biomass production was analyzed during drought stress with the use of lines differing only in the presence of fructans. Fructan-producing tobacco plants performed significantly better under polyethylene-glycol-mediated drought stress than wild-type tobacco. The growth rate of the transgenic plants was significantly higher (+55%), as were fresh weight (+33%) and dry weight (+59%) yields. The difference in weight was observed in all organs and was particularly pronounced in roots. Under unstressed control conditions the presence of fructans had no significant effect on growth rate and yield. Under all conditions the total nonstructural carbohydrate content was higher in the transgenic plants. We conclude that the introduction of fructans in this non-fructan-producing species mediates enhanced resistance to drought stress.

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.

A thylakoid processing protease is required for complete maturation of the lumen protein plastocyanin

Plastocyanin, a photosynthetic electron carrier functional in the chloroplast lumen, is synthesized in the cytosol as a precursor (preplastocyanin) with an amino-terminal transit sequence1. This transit peptide contains the information specifying import into and routing within the chloroplasts and is removed in at least two steps2. An intermediate is observed in the stroma after the transport of preplastocyanin through the chloroplast envelope; mature plastocyanin is present in the lumen, after transport over the thylakoid membrane. We show here that the stromal processing protease3 is not responsible for both processing events. It cleaves the precursor protein only to the intermediate size and a novel protease located in the thylakoids processes this intermediate to the mature protein. This second protease recognizes the processing intermediate but not the precursor. Thus plastocyanin import involves cleavage at the intermediate processing site mediated by the stromal protease and then cleavage at the mature processing site mediated by the thylakoid protease.

Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope

The outer membrane protein PupB of Pseudomonas putida WCS358 facilitates transport of iron complexed to the siderophores pseudobactin BN8 and pseudobactin BN7 into the cell. Its synthesis is induced by the presence of these specific siderophores under iron limitation. The signal transduction pathway regulating siderophore‐dependent expression of pupB was shown to consist of two regulatory proteins, PupI and PupR, and the PupB receptor itself. Mutational analysis of the regulatory genes suggested that PupI acts as a positive regulator of pupB transcription, whereas PupR modifies PupI activity dependent on the presence of pseudobactin BN8. PupI and PupR do not share homology with the classical bacterial two‐component systems but display significant similarity to the FecI and FecR proteins of Escherichia coli involved in regulation of ferric dicitrate transport. The function of the PupB receptor in pupB regulation was studied by the use of chimeric receptor proteins composed of PupB and the ferric pseudobactin 358 receptor PupA. This experiment revealed that PupB is involved in the initiation of the signal transduction pathway, implying a so far unique role for an outer membrane protein in signal transduction.

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants.

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.

The role of siderophores in potato tuber yield increase by Pseudomonas putida in a short rotation of potato

The effect of treatment of potato seed tubers withPseudomonas putida isolate WCS358 on tuber yield was studied in different crop rotations at the Experimental Farm ‘De Schreef’, near Lelystad. With untreated, tuber yield in a 1:3 (short) rotation compared to yield in a 1:6 (long) rotation of potato was decreased by 11% at 86 days (seed tuber harvest) and by 14% at 130 days (ware potato harvest) after seeding. Seed tuber treatment with the wild-type isolate WCS358 increased tuber yield with 13% in a short rotation of potato 86 days after seeding, whereas a siderophore-negative Tn5 transposon mutant of this isolate had no effect on tuber yield. Seed tuber treatment with the wild-type isolate or the siderophore-negative mutant in a long rotation of potato had no effect on tuber yield. At 130 days after seeding no effect of any of the seed tuber treatments was found in both short and long rotations of potato.Root colonization by siderophore-producing Tn5 transposon mutants of WCS358 was decreased at the end of the growing season. No difference in root colonization between siderophore-producing and siderophore-negative Tn5 transposon mutants was found at 130 days after seeding.Siderophore production seems to be a prerequisite in potato tuber yield increase by WCS358 under field conditions. This is the first time that the involvement of siderophores in growth stimulation has been demonstrated in the field.SamenvattingDe invloed van een behandeling van aardappelpootgoed metPseudomonas putida isolaat WCS358 op de knolopbrengst werd onderzocht in verschillende gewasrotaties on een proefveld van proefboerderij ‘De Schreef’, Flevopolder. In de controlebehandelingen werd in een nauwe aardappelrotatie (1:3) een reductie van 11% in opbrengst van pootaardappelen (86 dagen na het poten) geconstateerd ten opzichte van een ruime aardappelrotatie (1:6); 130 dagen na het poten werd een vermindering met 14% gevonden in de opbrengst van consumptieaardappelen.Pootgoedbehandeling met het siderofoorproducerende isolaat WCS358 verhoogde de opbrengst van pootaardappelen in de 1:3-rotatie met 13%. Een Tn5-transposonmutant van dit isolaat die het vermogen sideroforen te produceren had verloren, had geen effect op de opbrengst. In de 1:6-rotatie had behandeling van pootgoed met WCS358 geen effect op de opbrengst van pootaardappelen.Zowel in de nauwe (1:3) als in de ruimte (1:6) rotatie werd (130 dagen na het poten), geen effect van behandeling van pootgoed met WCS358 op de opbrengst van consumptieaardappelen gevonden.Wortelkolonisatie door siderofoorproducerende Tn5-transposonmutanten van WCS358 nam aan het eind van het seizoen af. Er werd, 130 dagen na het poten, geen verschil in wortelkolonisatie geconstateerd tussen siderofoorproducerende en siderofoornegatieve Tn5-transposonmutanten.Siderofoorproduktie blijkt een voorwaarde te zijn voor verhoging van de knolopbrengst door WCS358 onder veldomstandigheden. De verhoging van de knolopbrengst treedt alleen op in de nauwe aardappelrotatie. Dit is de eerste keer dat de betrokkenheid van sideroforen bij groeistimulatie onder veldomstandigheden is aangetoond.

Protein transport into and within chloroplasts

The chloroplast is a complex organelle which carries out a wide range of metabolic processes such as light capture and the biosynthesis of carbohydrates, fatty acid and amino acids. This organelle consists of three separate membrane systems which enclose three distinct soluble phases. Most of the chloroplast proteins are imported from the cytosol and directed into the six different compartments. This import and intraorganellar sorting process makes the chloroplast an interesting and promising system for the analysis of how proteins interact with and are translocated across biological membranes.