Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (GlnVal-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (K d ¼ 4.0 · 10 )2 lM, at pH 7.0 and 25°C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.

In addition to its role in protein synthesis and the plant cells' response to 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.

The recent progresses in the research on proline will be described, focusing on plants and covering proline metabolism and signal transduction as well as the role of this imino acid in stress response. Furthermore, the recently described developmental role of proline in flowering and reproduction will be illustrated and discussed.

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.

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 thaliana AtP5CS1 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.

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.

The plant oncogene rolB from Agrobacterium rhizogenes elicits differentiation and growth of neoplastic roots (hairy roots) in dicotyledonous plants. rolB-transformed plant cells show a sensitivity to auxin several order of magnitude higher than normal, and an increased binding capability to this most studied plant growth regulator. The oncogene rolB may thus represent an important tool in elucidating the still elusive mechanism of auxin signal perception/transduction and in shedding light on the role of this plant hormone in the control of plant growth and differentiation. So far, however, all attempts to clarify the biochemical activity and subcellular localization of the rolB gene product have been inconclusive. Here we show that the RolB protein overproduced in Escherichia coli has protein tyrosine phosphatase activity, and that, in transformed plant cells, is localised on the plasma membrane