Proline affects the size of the root meristematic zone in Arabidopsis

We reported previously that root elongation in Arabidopsis is promoted by exogenous proline, raising the possibility that this amino acid may modulate root growth. To evaluate this hypothesis we used a combination of genetic, pharmacological and molecular analyses, and showed that proline specifically affects root growth by modulating the size of the root meristem. The effects of proline on meristem size are parallel to, and independent from, hormonal pathways, and do not involve the expression of genes controlling cell differentiation at the transition zone. On the contrary, proline appears to control cell division in early stages of postembryonic root development, as shown by the expression of the G2/M-specific CYCLINB1;1 (CYCB1;1) gene. The overall data suggest that proline can modulate the size of root meristematic zone in Arabidopsis likely controlling cell division and, in turn, the ratio between cell division and cell differentiation

Proline synthesis in developing microspores is required for pollen development and fertility

Background: In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development. Results: We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by β-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility. Conclusions: Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains

Proline Affects Root Meristem Size In Arabidopsis thaliana

La crescita della radice in Arabidopsis thaliana si basa sull'attività della zona meristematica. La dimensione del meristema della radice è regolato dai fitoormoni auxina, citochinine e gibberelline, che controllano un circuito di espressione genica che converge sul gene SHY2. Regolando SHY2, gli ormoni vegetali controllano il rapporto tra la divisione cellulare e il differnziamento cellulare. In un lavoro precedente abbiamo riportato che l'allungamento della radice di Arabidopsis è promosso da concentrazioni micromolari di prolina esogena. Al fine di verificare se un mutante con deficit di prolina è caratterizzato da un problema nella crescita delle radici, abbiamo analizzato la lunghezza, rispetto al wild type , delle radici nel mutante p5cs1 p5cs2 / P5CS2. Le nostre analisi mostrano che piante con genotipo p5cs1 p5cs2 / P5CS2 sono caratterizzate da radici più brevi, e meristemi più piccole rispetto al wild type. Questi dati preliminari suggeriscono che la prolina in grado di modulare le dimensioni del meristema radicale e, a sua volta, la crescita delle radici. Per valutare questa ipotesi abbiamo usato una combinazione di analisi genetiche, farmacologiche e molecolari, e dimostrato che prolina colpisce specificamente crescita delle radici modulandone la dimensione del meristema . Gli effetti della prolina sulla dimensione del meristema sono paralleli e indipendenti dalle vie ormonali, e non comportano l'espressione di geni che controllano il differenziamento cellulare in corrispondenza della zona di transizione (SHY2). Al contrario, la prolina sembra controllare la divisione cellulare nelle prime fasi postembrionali di sviluppo delle radici, come indicato dall'espressione del gene CYCLINB1 che conrolla la transizione tra le fasi G2 / M. I dati complessivi indicano che la prolina può modulare la dimensione della zona meristematica della radice, probabilmente controllando la divisione cellulare.The Arabidopsis thaliana root growt relies on the activity of the root meristematic zone . The size of root meristem is regulated by the plant hormones auxin, cytokinin and gibberellin, which control a short regulatory circuit converging on the gene SHY2. By regulating SHY2, plant hormones control the ratio between cell division and cell differentiation. In a previus work we reported that root elongation in Arabidopsis is promoted by micromolar concentrations of exogenous proline. In order to verify whether a proline-deficient mutant is hampered in root growth, we analyzed the length, relative to wild type, of roots from the proline-deficient mutant p5cs1 p5cs2/P5CS2. Our analyses display that the p5cs1 p5cs2/P5CS2 mutant is characterized by shorter roots, and smaller meristems than wild type. These preliminary data suggest that proline can modulate root meristem size and, in turn, root growth. To evaluate this hypothesis we used a combination of genetic, pharmacological and molecular analyses, and showed that proline specifically affects root growth by modulating the size of the root meristem. The effects of proline on meristem size are parallel to, and independent from, hormonal pathways, and do not involve the expression of genes controlling cell differentiation at the transition zone (SHY2). On the contrary, proline appears to control cell division in early stages of postembryonic root development, as shown by the expression of the G2/M-specific CYCLINB1;1 gene and the G1/S-specific CYCLIND3;1 gene The overall data suggest that proline can modulate the size of root meristematic zone in Arabidopsis likely controlling cell division and, in turn, the ratio between cell division and cell differentiation

Role of Glutamate-Derived Amino Acids under Stress Conditions: The Case of Glutamine and Proline

A number of metabolic adaptive processes have been evolved in higher plants to cope with environmental stresses. Being sessile organisms unable to escape stressful conditions, plant survival is critically dependent by the rapid establishment of metabolic and physiologic responses that collectively attenuate or block the harmful effects of environmental injuries. Drought, cold and salt stress, in particular, are major threats for plant life, and cause, on a worldwide scale, dramatic losses in crop yields, especially in many developing countries and arid or semi-arid regions. The amino acids derived from glutamic acid, such as proline, γ-amino butyric acid (GABA), glutamine, arginine, as well as their close derivatives polyamines and nitric oxide (NO), play a special role in the metabolic adaptations of plants during abiotic stress. Glutamate itself seems to be not directly affected by stress conditions, but in reality plays an essential role as a donor of stress-related amino acids and metabolites as well as signal molecule involved in stress responses. Indeed most amino acids belonging to the glutamate family accumulate in large amounts under stress conditions and are thought to improve plant stress tolerance, although the molecular mechanisms through which these amino acids exert their action are still poorly understood. The study of the molecular and biochemical mechanisms by which this class of amino acids can improve plant tolerance to environmental stresses is of paramount importance in basic biology and can be regarded as a preliminary step towards the development of stress-tolerant and high-yield crop plants. The development of stress-tolerant crops of high productivity is a major issue in current research because of the increasing food demand of a growing world population, combined with the harmful effects of global warming, particularly drought, salinity and extreme temperatures. In this chapter the current state of the art of the glutamate-derived amino acids under stress conditions will be thoroughly revised and critically discussed with particular attention to the role of glutamine and proline for possible amelioration of yield and stress tolerance of agricultural crops

Proline is required for male gametophyte development in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>In crosses between the proline-deficient mutant homozygous for <it>p5cs1</it> and heterozygous for <it>p5cs2 (p5cs1 p5cs2/P5CS2)</it>, used as male, and different Arabidopsis mutants, used as females, the <it>p5cs2</it> mutant allele was rarely transmitted to the outcrossed progeny, suggesting that the fertility of the male gametophyte carrying mutations in both <it>P5CS1</it> and <it>P5CS2</it> is severely compromised.</p> <p>Results</p> <p>To confirm the fertility defects of pollen from <it>p5cs1 p5cs2/P5CS2</it> mutants, transmission of mutant alleles through pollen was tested in two ways. First, the number of progeny inheriting a dominant sulfadiazine resistance marker linked to <it>p5cs2</it> was determined. Second, the number of <it>p5cs2/p5cs2</it> embryos was determined. A ratio of resistant to susceptible plantlets close to 50%, and the absence of aborted embryos were consistent with the hypothesis that the male gametophyte carrying both <it>p5cs1</it> and <it>p5cs2</it> alleles is rarely transmitted to the offspring. In addition, in reciprocal crosses with wild type, about 50% of the <it>p5cs2</it> mutant alleles were transmitted to the sporophytic generation when <it>p5cs1 p5cs2/P5CS2</it> was used as a female, while less than 1% of the <it>p5cs2</it> alleles could be transmitted to the outcrossed progeny when <it>p5cs1 p5cs2/P5CS2</it> was used as a male. Morphological and functional analysis of mutant pollen revealed a population of small, degenerated, and unviable pollen grains, indicating that the mutant homozygous for <it>p5cs1</it> and heterozygous for <it>p5cs2</it> is impaired in pollen development, and suggesting a role for proline in male gametophyte development. Consistent with these findings, we found that pollen from <it>p5cs1</it> homozygous mutants, display defects similar to, but less pronounced than pollen from <it>p5cs1 p5cs2/P5CS2</it> mutants. Finally, we show that pollen from <it>p5cs1 p5cs2/P5CS2</it> plants contains less proline than wild type and that exogenous proline supplied from the beginning of another development can partially complement both morphological and functional pollen defects.</p> <p>Conclusions</p> <p>Our data show that the development of the male gametophyte carrying mutations in both <it>P5CS1</it> and <it>P5CS2</it> is severely compromised, and indicate that proline is required for pollen development and transmission.</p

Role of proline and GABA in sexual reproduction of angiosperms

Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance.publishe

Anthrax Protective Antigen Retargeted with Single‐Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.NSF (Grant CHE-1351807