Elsevier

Plant Science

Volume 181, Issue 2, August 2011, Pages 140-150
Plant Science

Elevation of free proline and proline-rich protein levels by simultaneous manipulations of proline biosynthesis and degradation in plants

https://doi.org/10.1016/j.plantsci.2011.04.013Get rights and content

Abstract

Proline-rich proteins (PRP) are cell wall and plasma membrane-anchored factors involved in cell wall maintenance and its stress-induced fortification. Here we compare the synthesis of P5C as the proline (Pro) precursor in the cytosol and chloroplast by an introduced alien system and evaluate correlation between PRP synthesis and free Pro accumulation in plants.

We developed a Pro over-producing system by generating transgenic tobacco plants overexpressing E. coli P5C biosynthetic enzymes; Pro-indifferent gamma-glutamyl kinase 74 (GK74) and gamma-glutamylphosphate reductase (GPR), as well as antisensing proline dehydrogenase (ProDH) transcription. GK74 and GPR enzymes were targeted either to the cytosol or plastids. Molecular analyses indicated that the two bacterial enzymes are efficiently expressed in plant cells, correctly targeted to the cytosol or chloroplasts, and processed to active enzymatic complexes in the two compartments. Maximal Pro increase is obtained when GK74 and GPR are active in chloroplasts, and ProDH mRNA level is reduced by anti-sense silencing, resulting in more than 50-fold higher Pro content compared to that of wild type tobacco plants. The Pro over-producing system efficiently works in tobacco and Arabidopsis. The elevation of Pro levels promotes accumulation of ectopically expressed Cell Wall Linker Protein (AtCWLP), a membrane protein with an external Pro-rich domain. These results suggest that the Pro-generating system can support endogenous or alien PRP production in plants.

Highlights

► The study describes an alien system that increases proline content in plant cells. ► This system leads to more than 50-fold proline accumulation in transgenic plants. ► It supports the synthesis of a proline-rich protein (PRP), which has been chosen as a representative of PRPs whose assembly in the plasma membrane and cell wall may increase tolerance to certain stresses.

Introduction

Free proline (Pro) accumulates in plants in response to a wide range of environmental stresses including water deprivation, salinity, low and high temperatures, pathogen infection, heavy metal toxicity, anaerobiosis, nutrient deficiency, atmospheric pollution, and UV irradiation [1], [2], [3]. The role of high Pro levels in combating adverse environmental effects is still under debate. Most interpretations that support Pro contribution to tolerance against environmental stresses rely on the ability of Pro to mediate osmotic adjustment, stabilization of subcellular structures, scavenging of free radicals, and its involvement in shuttling chemical energy required for the stress recovery process [1], [2], [3].

In E. coli, the first two steps in the main pathway of Pro synthesis from glutamate (Fig. 1) are catalyzed by gamma-glutamyl kinase (GK), encoded by proB, and gamma-glutamylphosphate reductase (GPR or GSD), encoded by proA, which together form a stable enzymatic complex. The delta-pyrroline-5-carboxylate (P5C) product formed by the GK–GPR complex (Fig. 1), is immediately reduced to Pro by delta-pyrroline-5-carboxylate reductase (P5CR), encoded by proC. GK, the key enzyme in the biosynthesis route, is feedback regulated by Pro. Therefore, Pro accumulation does not occur in wild-type E. coli cells. The mutant E. coli proB74 gene encodes a modified GK enzyme (denoted as GK74), which is insensitive to Pro up to 100 mM, and can therefore drive Pro synthesis in the presence of high cellular Pro concentration. Increased Pro production and osmotolerance in bacteria have been observed in mutants expressing the proB74 gene [4], [5].

Pro is produced from either glutamate or ornithine (Fig. 1) in young Arabidopsis plants, whereas in mature plants or during exposure to stress the glutamate pathway usually dominates [6]. The bifunctional enzyme, delta-pyrroline-5-carboxylate synthase (P5CS, Fig. 1), catalyses the first two steps of P5C formation. It shows amino acid homology to conserved domains of bacterial GK and GPR enzymes [7], [8], [9], [10]. The available P5CS cDNA sequences do not contain a recognizable sequence of plastid-targeting transit-peptide, and therefore P5CS was assumed to be a cytosolic enzyme. However, recently the Arabidopsis P5CS1 and P5CS2 cDNAs were separately fused to the GFP coding sequence and when expressed in transgenic Arabidopsis plants, P5CS1–GFP accumulated in the chloroplasts during salt and osmotic stresses [11]. Under normal growth conditions, P5CS is feedback-regulated and its activity is completely blocked by 10 mM Pro [9], whereas during stress imposition the cellular levels of Pro may exceed 100 mM with no inhibitory effect. Ectopic expression of Vigna P5CS cDNA in tobacco resulted in 18-fold elevation of free Pro content under normal growth conditions, and increased tolerance to salt stress [12], despite the sensitivity of the enzyme to low Pro concentrations. However, the osmotic adjustment measurements performed in this study [12] have been critically discussed [13]. Mutated Vigna P5CS, with relative insensitivity to free Pro, was obtained by site-directed mutagenesis [14]. The mutated P5CS could further increase Pro accumulation by two-fold upon being constitutively expressed in transgenic tobacco seedlings under normal or high-salt (200 mM) conditions [15]. Nonetheless, the presence of an inhibitor in roots that inactivates P5CS [14] may negatively affect the activity of endogenous and ectopic P5CSs in roots, the primary sites of confronting salinity stress.

P5CR, which catalyses Pro formation from P5C, is encoded by a single gene in Arabidopsis and is believed to be a cytosolic enzyme. However, cell fractionation studies could detect P5CR activity in both cytosol and chloroplast [16]. Thus, the findings that P5CS1 [11] and P5CR are probably present in chloroplasts suggest that Pro synthesis might also occur in the chloroplast, although this assumption still awaits biochemical verification. Overexpression of ectopic P5CR in transgenic plants does not affect proline levels, therefore, P5CR is not considered as a candidate for ectopic expression aiming at an increase of free Pro levels in plants [16]. However, it is not expected to be a limiting factor when high levels of P5C are produced, since increased Pro levels were obtained in tobacco plants overexpressing the Vigna P5CS mutant, which is not feed-back inhibited by Pro [14], [15].

In plants, oxidative degradation of proline to glutamate is carried out in the mitochondria by sequential actions of proline dehydrogenase (ProDH) and P5C-dehydrogenase (P5CDH) (Fig. 1). ProDH1 cDNA was first isolated from Arabidopsis by Kiyosue et al. [17]. There are two highly homologous ProDH isozymes in Arabidopsis and alfalfa [18], [19]. ProDH is a FAD-enzyme, which is bound to the matrix side of mitochondrial inner membrane and transfers electrons directly to the mitochondrial electron transport chain [20]. ProDH and P5CR comprise the Pro-P5C cycle, which oxidizes proline to P5C in mitochondria and reduces P5C to Pro in the cytosol, thereby maintaining proline and P5C homeostasis (see below). P5CDH, the second enzyme in Pro catabolism (Fig. 1), catalyses the oxidation of P5C to glutamate [21], a process that together with the Pro-P5C cycle controls P5C levels and thereby prevents ROS formation [20]. The enzymatic activities of ProDH and P5CDH in vitro are negatively affected by high concentrations of Cl anions, while in intact cells no conspicuous Cl inhibition of P5CDH could be recorded [22], [23].

Analyses of transcription during abiotic stress and subsequent recovery showed that the levels of P5CS transcripts are elevated during stress (Appendix, Fig. A1) and gradually decrease during the post-stress period [8], [10], [22], [23], [24]. In Arabidopsis, the two P5CS1 and P5CS2 genes are differently expressed, whereas P5CS1 expression is upregulated by abiotic stresses and ABA [11], [25], P5CS2 acts as a housekeeping enzyme, being more expressed in meristematic tissues of vegetative and reproductive organs [11], [25]. Conversely, transcript levels of ProDH are gradually reduced within several hours of abiotic stress (Fig. A1), and rapidly increased upon relief from stress [19], [23], [24], [26], [27], [28], [29]. Thus, a reciprocal regulation of P5CS and ProDH genes appears to be the key mechanism in controlling Pro levels under abiotic stress conditions.

The role of high levels of Pro in rendering plants more tolerant to salinity, drought and other osmotic stresses has been extensively discussed [3]. However, the need for free Pro to support the synthesis of proline-rich proteins (PRPs), has drawn less attention. Upon constitutive anti-sensing of P5CS1 and P5CS2 in Arabidopsis, Nanjo et al. [30] observed a significant decrease in free Pro in the cytosol accompanied by a reduction of Pro and hydroxyproline content in the cell wall, suggesting less accumulation of cell wall PRPs. The plants with P5CS antisensing showed decreased tolerance to osmotic stress and abnormal development of shoot tissues [30]. This observation provided the first evidence that linked cell wall PRP content with proline levels and stress sensitivity. Many of the cell wall proteins are PRPs. In most of them Pro residues are arranged in repetitive motifs which also contain hydroxyproline. Pro hydroxylation and sequential glycosylation occur in the ER and Golgi network before the assembly as plasma membrane components or secretion to the cell wall [31]. Most PRPs are involved in cell wall assembly and maintenance during growth and development. Two PRP subfamilies (extensins and P/HRGPs) play important roles in plant tolerance to biotic and abiotic stresses, due to their stress-induced oxidation, which forms inter- and intra-molecular cross-links that strengthen the cell wall structure [33]. The 8CM-HyPRP proteins comprise another type of PRPs that are anchored to the plasma membrane by three conserved trans-membrane domains, characterized by a typical and conserved arrangement of eight cysteine residues. Some of these proteins are induced by low temperature and are involved in increased tolerance to abiotic stresses [34], [35].

Considering the recently raised hypothesis of Pro synthesis in the chloroplast [3], in the present study we compare the ability of plant cells to over-produce Pro, when the key step of synthesizing P5C (Fig. 1) is performed either in the cytosol or chloroplast. A versatile ectopic system comprising of E. coli proA and the Pro-indifferent proB74 mutated gene, was introduced into the nuclear genome of tobacco and Arabidopsis. The constitutively expressed bacterial enzymes were targeted to the cytosol or chloroplast. We show that the highest free Pro levels are obtained when P5C synthesis is performed in the chloroplast and the expression of the endogenous ProDH is anti-sensed. This high free Pro accumulation promotes PRP production. By co-expressing the Arabidopsis cell wall-plasma membrane linker protein (CWLP) [36], from the 8CM-HyPRP family as a PRP model protein, we could show that the synthesis of the CWLP is substantially enhanced when the proline-overproducing system is active in chloroplasts resulting in increased tolerance to a short heat shock. Hence, this approach may provide a system for enhancing the synthesis of proline-rich proteins in plants for either increasing the cell wall defense potential or producing biotechnologically important therapeutic proteins, such as collagen [37].

Section snippets

Plants and growth conditions

Nicotiana tabacum cv. Samsun (NN) and Arabidopsis thaliana cv. Columbia (Col-0) were grown at 25 or 22 °C, respectively, in a 16 h light/8 h dark regime, at light intensity of 100 μmol cm−2 s−1.

GK and GPR enzyme assay

The combined assay was carried out by over-expressing either wild type bacterial GK encoded by proB (X00786) or the mutant version GK74 encoded by proB74 [4], along with GPR (GSD) encoded by proA in E. coli (Fig. 1). The GK–GPR coupled assay was performed in vitro according to Smith et al. [38].

Formation of translational fusions proB74 and proA with the sequence of the transit peptide of RUBISCO small subunit

The 5′ sequence

Characterization of the Pro-insensitive enzyme GK74

To obtain Pro over-production in plant cells using the bacterial GK and GPR (Fig. 1) the mutant E. coli proB74 gene encoding the GK74 enzyme [4], was examined for its capacity to maintain enzymatic activity in the presence of high Pro concentrations in vitro. Wild type GK and the GK74 mutant were overexpressed along with GPR in E. coli and GK–GPR coupled assays were performed in vitro (Appendix, Fig. A2). These assays confirmed the findings of Smith [44] that GK74 is two orders of magnitude

Discussion

Increase in free cellular Pro during various abiotic stresses has been extensively discussed, attributing a multi-functional protective role to this evolutionary maintained phenomenon found in most plant species [1], [3]. In this study, we examined the possible synthesis of the Pro precursor P5C in chloroplasts, and the linkage between the availability of free cellular Pro and the synthesis of proline-rich-proteins. PRP proteins are playing important roles in cell wall development and its

Acknowledgements

The constructive discussions with Dr. E. Sadot (The Volcani Institute, Bet Dagan, Israel) and Prof. P. Goloubinoff (Université de Lausanne, Lausanne, Switzerland) are highly appreciated. This work was supported by the European Union FP5 grant (OLRT-2001-00841) (C.K., L.S and A.Z.) and NIH R01-GM3199994 (LNC). C.K., L.S. and A.Z. are members of the EU COST action FA0605 INPAS that inspired this issue.

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    Present address: FuturaGene Ltd., P.O. Box 199, 2 Pekeris Street, Park Tamar, Rehovot 76100, Israel.

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    Present address: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.

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