Roberto Mattioli

Proline accumulation in plants: not only stress.

In addition to its role in protein synthesis, and in the response of plant cell to a number of environmental stresses, circumstantial evidence suggest that proline may also play a role in flowering and development both as a metabolite and as a signal molecule. Although there is a growing consensus that proline is of special importance throughout the reproductive phase, from flower transition to seed development, a general agreement on the molecular and genetic mechanisms proline is involved in, is yet to be established. In this paper we shall review and critically discuss most of the evidence supporting a role for proline in plant development, paying special attention to the recently reported role of proline in flower transition.

Modulation of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis.

We reported previously that the plant oncogene rolD anticipates and stimulates flowering in Nicotiana tabacum, and encodes ornithine cyclodeaminase, an enzyme catalysing the conversion of ornithine to proline. To investigate on the possible role of proline in flowering, we altered the expression of AtP5CS1, encoding the rate-limiting enzyme of proline biosynthesis in plants. Accordingly we characterized a mutant line containing a T-DNA insertion into AtP5CS1 and introduced in Arabidopsis thalianaAtP5CS1 under the control of the CaMV35S promoter. As expected homozygous p5cs1 mutants behaved as late flowering. In addition p5cs1 mutants exhibited a shorter size and contained lower levels of proline, compared to wild type. 35S-P5CS1 plants, manifested, early in development, overexpression of P5CS1 and accumulation of proline, leading to early flowering, both under long- and short-day conditions. Later in development, down-regulation of P5CS1 occurred in 35S-P5CS1 leaves, leading to proline reduction, and, in turn, impaired bolting and stunted growth. Salt-stress restored expression of P5CS1 and proline accumulation in P5CS1-transformed plants, as well as rescuing growth. Our data suggest that proline plays a key role in flower transition, bolting and coflorescence formation.

The proline biosynthetic genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition but not in embryo development

Overexpression of the proline biosynthetic gene P5CS1 results in early flowering in Arabidopsis. However, the p5cs1 loss-of-function mutant exhibits a modest delay in flowering, suggesting that P5CS2, a duplicated P5CS1 gene present in the Arabidopsis, may also play a role in flower transition. In situ mRNA hybridizations and quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that P5CS1 and P5CS2 are expressed at similar levels and with the same pattern of expression in vegetative and floral shoot apical meristems as well as in axillary meristems. Arabidopsis lines homozygous for the p5cs1 mutant and simultaneously heterozygous for the p5cs2 mutation showed a stronger late-flowering phenotype than p5cs1 single mutants, confirming that also P5CS2 plays a role in flower transition and supporting the notion of overlapping functions of the two P5CS genes in this developmental process. P5CS1 and P5CS2 have identical messenger RNA (mRNA) distributions also in embryos, but only p5cs2 mutant embryos exhibit alterations of the cellular division planes and consequently stop developing. This suggests a specific role of P5CS2 in embryogenesis and an involvement of proline in cell division. Accordingly, exogenous proline accelerated organ growth and meristem formation, and stimulated expression of the cell cycle-related protein CYCB1;1.

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.

Proline is required for male gametophyte development in Arabidopsis

BackgroundIn crosses between the proline-deficient mutant homozygous for p5cs1 and heterozygous for p5cs2 (p5cs1 p5cs2/P5CS2), used as male, and different Arabidopsis mutants, used as females, the p5cs2 mutant allele was rarely transmitted to the outcrossed progeny, suggesting that the fertility of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised.ResultsTo confirm the fertility defects of pollen from p5cs1 p5cs2/P5CS2 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 p5cs2 was determined. Second, the number of p5cs2/p5cs2 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 p5cs1 and p5cs2 alleles is rarely transmitted to the offspring. In addition, in reciprocal crosses with wild type, about 50% of the p5cs2 mutant alleles were transmitted to the sporophytic generation when p5cs1 p5cs2/P5CS2 was used as a female, while less than 1% of the p5cs2 alleles could be transmitted to the outcrossed progeny when p5cs1 p5cs2/P5CS2 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 p5cs1 and heterozygous for p5cs2 is impaired in pollen development, and suggesting a role for proline in male gametophyte development. Consistent with these findings, we found that pollen from p5cs1 homozygous mutants, display defects similar to, but less pronounced than pollen from p5cs1 p5cs2/P5CS2 mutants. Finally, we show that pollen from p5cs1 p5cs2/P5CS2 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.ConclusionsOur data show that the development of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised, and indicate that proline is required for pollen development and transmission.

Protective effects of Brassica oleracea sprouts extract toward renal damage in high-salt-fed SHRSP: role of AMPK/PPARa/UCP2 axis

Objectives: Renal damage precedes occurrence of stroke in high-sodium/low-potassium-fed stroke-prone spontaneously hypertensive rat (SHRSP). We previously reported a marked suppression of uncoupling protein-2 (UCP2) upon high-salt Japanese-style diet in SHRSP kidneys. Vegetable compounds are known to exert protective effects in cardiovascular diseases. We aimed at evaluating the impact of Brassica oleracea sprouts juice toward renal damage in Japanese diet-fed SHRSP and exploring the role of 5′-adenosine monophosphate-activated protein kinase (AMPK)/NAD-dependent deacetylase sirtuin-1 (SIRT1)/peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; (PGC1&agr;)/peroxisome proliferator-activated receptor-&agr; (PPAR&agr;)/UCP2 axis. Methods: SHRSP received Japanese diet for 4 weeks. A group of SHRSP received Japanese diet and B. oleracea. A third group received Japanese diet, B. oleracea, and PPAR&agr; inhibitor (GW6471). A group of SHRSP fed with regular diet served as control. Results: Japanese diet induced marked increases of oxidative stress, inflammation, and proteinuria, along with glomerular and tubular damage, as compared with regular diet. A significant suppression of AMPK/UCP2 pathway was observed. Despite Japanese diet feeding, concomitant administration of B. oleracea prevented oxidative stress accumulation, inflammation, renal damage, and proteinuria. All components of the UCP2 regulatory pathway were significantly increased by B. oleracea. Superoxide dismutase 2 and phosphoendothelial nitric oxide synthase were also stimulated. Addition of PPAR&agr; inhibitor to B. oleracea and Japanese diet significantly reduced the B. oleracea beneficial effects. SBP levels were comparable among the different groups of rats. In vitro, UCP2 inhibition by genipin offset the antioxidant effect of B. oleracea in renal mesangial and proximal tubular cells. Conclusion: B. oleracea administration prevented renal damage in salt-loaded SHRSP, independently from SBP, with parallel stimulation of AMPK/SIRT1/PGC1&agr;/PPAR&agr;/UCP2 axis. Stimulation of the latter mechanism may provide relevant renal protective effect and play a therapeutic role in target organ damage progression in hypertension.

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.

Proline affects the size of the root meristematic zone in Arabidopsis

BackgroundWe reported previously that root elongation in Arabidopsis is promoted by exogenous proline, raising the possibility that this amino acid may modulate root growth.ResultsTo 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.ConclusionsThe 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.

Neuroprotective Effect of Brassica oleracea Sprouts Crude Juice in a Cellular Model of Alzheimer's Disease.

β-Amyloid peptide (Aβ) aberrant production and aggregation are major factors implicated in the pathogenesis of Alzheimers disease (AD), causing neuronal death via oxidative stress. Several studies have highlighted the importance of polyphenolic antioxidant compounds in the treatment of AD, but complex food matrices, characterized by a different relative content of these phytochemicals, have been neglected. In the present study, we analyzed the protective effect on SH-SY5Y cells treated with the fragment Aβ 25–35 by two crude juices of broccoli sprouts containing different amounts of phenolic compounds as a result of different growth conditions. Both juices protected against Aβ-induced cytotoxicity and apoptotic cell death as evidenced by cell viability, nuclear chromatin condensation, and apoptotic body formation measurements. These effects were mediated by the modulation of the mitochondrial function and of the HSP70 gene transcription and expression. Furthermore, the juices upregulated the intracellular glutathione content and mRNA levels or activity of antioxidant enzymes such as heme oxygenase-1, thioredoxin, thioredoxin reductase, and NAD(P)H:quinone oxidoreductase 1 via activation of NF-E2-related factor 2 (Nrf2). Although the effects of the two juices were similar, the juice enriched in phenolic compounds showed a greater efficacy in inducing the activation of the Nrf2 signalling pathway.