Nighttime sugar starvation orchestrates gibberellin biosynthesis and plant growth in Arabidopsis.

A plant's eventual size depends on the integration of its genetic program with environmental cues, which vary on a daily basis. Both efficient carbon metabolism and the plant hormone gibberellin are required to guarantee optimal plant growth. Yet, little is known about the interplay between carbon metabolism and gibberellins that modulates plant growth. Here, we show that sugar starvation in Arabidopsis thaliana arising from inefficient starch metabolism at night strongly reduces the expression of ent-kaurene synthase, a key regulatory enzyme for gibberellin synthesis, the following day. Our results demonstrate that plants integrate the efficiency of photosynthesis over a period of days, which is transduced into a daily rate of gibberellin biosynthesis. This enables a plant to grow to a size that is compatible with its environment.

Influence of partial replacement of soya bean meal by faba beans or peas in heavy pigs diet on meat quality, residual anti-nutritional factors and phytoestrogen content.

The study evaluated the partial substitution of soybean meal by faba beans (18%) or peas (20%) as additional protein sources in diets destined for typical Italian heavy pig production. It compared animal performances, meat quality, the presence of residual anti-nutritional factors (ANF) and phytoestrogens in plasma and meat and the possible effects on pig health, by evaluating oxidative, inflammatory and pro-atherogenic markers. The results showed that the productive performances, expressed as body weight and feed conversion ratio, of pigs fed with faba bean and pea diets were similar to those of pigs fed only the soybean meal. Meat quality of pigs fed with the three diets was similar in colour, water-holding capacity, tenderness and chemical composition. Despite the higher levels of phytoestrogen in the plasma of pigs fed only the soybean meal, phytoestrogen concentration in the muscle was equivalent to that of animals fed diets with faba beans, whereas pigs fed a diet with peas showed a lower concentration. Inflammation and pro-atherogenic parameters did not show significant differences among the three diets. Overall, the partial substitution of soybean meal by faba beans appears more interesting than with peas, particularly in relation to the higher amount of polyphenols in the diet and the highest concentration of phytoestrogens found in the plasma and muscle of animals, while the pyrimidine anti-nutritional compounds present in the diet did not appear to accumulate and had no effect on the growth performance of animals.

Tomato fruit development in the auxin-resistant dgt mutant is induced by pollination but not by auxin treatment.

In tomato (Solanum lycopersicum Mill.), auxin is believed to play a pivotal role in controlling fruit-set and early ovary growth. In this paper we investigated the effect of the reduced auxin sensitivity exhibited by the diageotropica (dgt) tomato mutant on ovary growth during early stage of fruit development. Here we show that in hand-pollinated ovaries fruit-set was not affected by the dgt lesion while fruit growth was reduced. This reduction was associated with a smaller cell size of mesocarp cells, with a lower mean C values and with a lower gene expression of the expansin gene LeExp2. When a synthetic auxin (4-CPA, chlorophenoxyacetic acid) was applied to the flowers of wild type plants, parthenocarpic ovary growth was induced. On the contrary, auxin application to the flowers of dgt plants failed to induce parthenocarpy. Hand-pollinated ovaries of dgt contained higher levels of IAA compared to wild type and this was not associated with high transcript levels of genes encoding a key regulatory enzyme of IAA biosynthesis (ToFZYs) but with lower expression levels of GH3, a gene involved in the conjugation of IAA to amino acids. The expression of diverse Aux/IAA genes and SAUR (small auxin up-regulated RNA) was also altered in the dgt ovaries. The dgt lesion does not seem to affect specific Aux/IAA genes in terms of transcript occurrence but rather in terms of relative levels of expression. Transcript levels of Aux/IAA genes were up regulated in auxin-treated ovaries of wild-type but not in dgt. Together, our results suggest that dgt ovary cells are not able to sense and/or transduce the external auxin signal, whereas pollinated dgt ovary cells are able to detect the IAA present in fertilized ovules promoting fruit development.

Ethylene produced by the endosperm is involved in the regulation of nucellus programmed cell death in Sechium edule Sw.

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we analysed the role of the endosperm as main regulator of nucellus PCD. We demonstrated that endosperm produces high amount of ethylene, nitric oxide and indoleacetic acid. We examined the role of these small and diffusible signalling molecules in the regulation of nucellus PCD and we tried to elucidate how they can cooperate and regulate each other into the endosperm. We showed that ethylene acts a positive regulator of nucellus PCD and its synthesis can be in part induced by nitric oxide. High levels of IAA were detected both in the endosperm and in dying nucellus but this hormone is not directly involved in the execution of PCD.

Changes in ultrastructure, protease and caspase-like activities during flower senescence in Lilium longiflorum.

The last phase of flower development is senescence during which nutrients are recycled to developing tissues. The ultimate fate of petal cells is cell death. In this study we used the ethylene-insensitive Lilium longiflorum as a model system to characterize Lily flower senescence from the physiological, biochemical and ultrastructural point of view. Lily flower senescence is highly predictable: it starts three days after flower opening, before visible signs of wilting, and ends with the complete wilting of the corolla within 10 days. The earliest events in L. longiflorum senescence include a fall in fresh and dry weight, fragmentation of nuclear DNA and cellular disruption. Mesophyll cell degradation is associated with vacuole permeabilization and rupture. Protein degradation starts later, coincident with the first visible signs of tepal senescence. A fall in total protein is accompanied by a rise in total proteases, and also by a rise of three classes of caspase-like activity with activities against YVAD, DEVD and VEID. The timing of the appearance of these caspase-like activities argues against their involvement in the regulation of the early stages of senescence, but their possible role in the regulation of the final stages of senescence and cell death is discussed.

The extreme dwarf phenotype of the GA-sensitive mutant of sunflower, dwarf2, is generated by a deletion in the ent-kaurenoic acid oxidase1 (HaKAO1) gene sequence.

A dwarf mutant, dw arf 2 (dw2), was isolated from sunflower (Helianthus annuus). The most obvious alterations of dw2 plants were the lack of stem growth, reduced size of leaves, petioles and flower organs, retarded flower development. Pollen and ovules were produced but the filaments failed to extrude the anthers from the corolla. The dw2 phenotype was mainly because of reduced cell size. In dw2 leaves, the dark-green color was not so much due to higher pigment content, but was correlated with a changed leaf morphology. The mutant responded to the application of bioactive gibberellins (GAs). The levels of ent-7α-hydroxykaurenoic acid, GA(19), GA(20) and GA(1) in dw2 seedlings were severely decreased relative to those in its wild type (WT). ent-Kaurenoic acid was actively converted to ent-7α-hydroxykaurenoic acid in WT plants but quite poorly in dw2 plants. All together these data suggested that the dw2 mutation severely reduced the flux through the biosynthetic pathway leading to active GAs by hampering the conversion of ent-kaurenoic acid to GA(12). Two ent-kaurenoic acid oxidase (KAO) genes were identified. HaKAO1 was expressed everywhere in sunflower organs, while HaKAO2 was mainly expressed in roots. We demonstrated that a DNA deletion in HaKAO1 of dw2 generated aberrant mRNA-splicing, causing a premature stop codon in the amino acid sequence. In dw2 calli, Agrobacterium-mediated transfer of WT HaKAO1 cDNA restored the WT endogenous levels of GAs. In segregating BC(1) progeny, the deletion co-segregated with the dwarf phenotype. The deletion was generated near to a breakpoint of a more complex chromosome rearrangement.

Nitric oxide and hydrogen peroxide involvement during programmed cell death of Sechium edule nucellus.

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we show that nucellus PCD is accompanied by a considerable production of both nitric oxide and hydrogen peroxide (NO and H(2)O(2)). Interestingly, each of the two molecules is able to induce the production of the other and to cause cell death when applied to a living nucellus. We show that the induced cell death has features of a PCD, accompanied by profound changes in the morphology of the nuclei and by a massive degradation of nuclear DNA. Moreover, we report that NO and H(2)O(2) cause an induction of caspase-like proteases previously characterized in physiological nucellar PCD.

The involvement of indole-3-acetic acid in the control of stem elongation in dark- and light-grown pea (Pisum sativum) seedlings.

We investigated the role of auxin on stem elongation in pea (Pisum sativum L.) grown for 10d in continuous darkness or under low-irradiance blue, red, far red and white light. The third internode of treated seedlings was peeled and the tissues (epidermis and cortex+central cylinder) were separately analyzed for the concentration of free and conjugated indole-3-acetic acid (IAA). Under red, far red and white light internode elongation was linearly related with the free IAA content of all internode tissues, suggesting that phytochrome-dependent inhibition of stem growth may be mediated by a decrease of free IAA levels in pea seedlings. The correlation between IAA and internode elongation, however, did not hold for blue light-grown seedlings. The hypothesis that the growth response under low-irradiance blue light might be correlated with the lack of phytochrome B signalling and changes in gibberellin metabolism is discussed in view of current knowledge on hormonal control of stem growth.

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases.

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.

Tomato fruit set driven by pollination or by the parthenocarpic fruit allele are mediated by transcriptionally regulated gibberellin biosynthesis.

We investigated the role of gibberellins (GAs) in the phenotype of parthenocarpic fruit (pat), a recessive mutation conferring parthenocarpy in tomato (Solanum lycopersicum L.). Novel phenotypes that parallel those reported in plants repeatedly treated with gibberellic acid or having a GA-constitutive response indicate that the pat mutant probably expresses high levels of GA. The retained sensitivity to the GA-biosynthesis inhibitor paclobutrazol reveals that this condition is dependent on GA biosynthesis. Expression analysis of genes encoding key enzymes involved in GA biosynthesis shows that in normal tomato ovaries, the GA20ox1 transcript is in low copy number before anthesis and only pollination and fertilization increase its transcription levels and, thus, GA biosynthesis. In the unpollinated ovaries of the pat mutant, this mechanism is de-regulated and GA20ox1 is constitutively expressed, indicating that a high GA concentration could play a part in the parthenocarpic phenotype. The levels of endogenous GAs measured in the floral organs of the pat mutant support such a hypothesis. Collectively, the data indicate that transcriptional regulation of GA20ox1 mediates pollination-induced fruit set in tomato and that parthenocarpy in pat results from the mis-regulation of this mechanism. As genes involved in the control of GA synthesis (LeT6, LeT12 and LeCUC2) and response (SPY) are also altered in the pat ovary, it is suggested that the pat mutation affects a regulatory gene located upstream of the control of fruit set exerted by GAs.

DNA degradation during programmed cell death in Phaseolus coccineus suspensor.

The suspensor of Phaseolus coccineus L. degenerates at the cotyledonary stage of embryogenesis when it is no longer necessary for continued embryonic development; this degeneration is considered to be a typical example of the so-called developmental programmed cell death (PCD) in plants. The presence of specific hallmarks of PCD as it occurs during the degeneration of P. coccineus suspensor was investigated in the current study. By using the TUNEL assay and electrophoretic analysis, we found evidence of nuclear DNA degradation, a known feature of PCD, in the endosperm and degenerating suspensors. Degeneration of the suspensor begins after degeneration in the endosperm and it starts in the neck region, spreading basipetally towards the knob. We conclude from this study that suspensor degeneration in P. coccineus occurs by means of PCD and displays typical hallmarks of PCD, such as DNA fragmentation. PCD in the suspensor is a highly asynchronous process, originating first in the neck cells and subsequently spreading to the basal cells.

Are retinal and retinal-binding proteins involved in stomatal response to blue light?

Stomata respond to blue light and it is generally believed that the photoreceptor for this response is located inside the guard cells. Only a small number of blue light photoreceptors have been identified so far, namely cryptochromes and phototropins, and they show overlapping functions in regulating many different responses to light. The possibility that plants may possess other receptors regulating blue light responses under different light conditions cannot be excluded. In this paper we show the presence of two retinal-binding proteins in Commelina communis and we report the identification of retinal, a chromophore usually bound to the photoreceptor rhodopsin and previously identified in algae and other higher plants. We show that, under our experimental conditions, stomata open promptly when exposed to blue light and we demonstrated that this response is dependent on retinal. We hypothesise that rhodopsin-like retinal-binding proteins might be involved in stomatal response to blue light.

Expression of two genes encoding gibberellin 2- and 3-oxidases in developing seeds of Phaseolus coccineus.

We have isolated PcGA3ox1, a cDNA clone from developing runner bean (Phaseolus coccineus) seeds that shows significant amino acid homology with the gibberellin (GA) 3-oxidases. A recombinant fusion protein of PcGA3ox1 converted GA20 and GA9 to GA1 and GA4, respectively. In situ hybridization results showed that transcripts of this gene accumulate specifically within the suspensor of globular-stage embryos. PcGA3ox1 mRNA begins to accumulate in the epidermal cells of the embryo proper and is also detectable in the endosperm during the transition from globular- to heart-stage embryos. PcGA3ox1 transcripts were localized exclusively in the cotyledons from the early cotyledonary stage up to the cotyledonary stage. Transcripts of the previously cloned GA 2-oxidase (PcGA2ox1) from developing seeds of runner bean were found primarily within the suspensor neck region from the late globular stage up to the heart stage. PcGA2ox1 mRNA was detectable in the whole suspensor from the early cotyledonary stage, and was found in the inner layer of integuments at the cotyledonary stage. Soluble enzyme preparations made from suspensors and embryos at two stages of embryogenesis (the heart and cotyledonary stages) were incubated with [14C]GA20 and [14C]GA1. Only young suspensor preparations converted GA20 to GA1 and GA5. Both suspensor preparations converted GA1 to GA8. Both embryo preparations converted GA20 to GA1, but were unable to convert GA1 to GA8.

The metabolic basis for 2,4-D resistance in two variant cell lines of carrot.

In the present work, the characterization of two variant cell lines of carrot capable of growing in high (92 µmol L-1) concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. Both these cell lines (4w77 and 4w13) show a significantly lower uptake of 2,4-D with respect to wild-type (wt) cells. In contrast to wt cells, influx of IAA is not reduced by the addition of 100 µM 2,4-D and the presence of this compound appears to stimulate IAA uptake. When grown in the presence of high concentrations of 2,4-D, both 4w77 and 4w13 cells show behavioural differences: instead of lowering the endogenous level of free IAA, the two resistant lines react to the high exogenous concentrations of auxin by raising the level of the free hormone. In 4w77 cells, this is accomplished by reduction of auxin released in the external medium or converted to amide-linked conjugates. In 4w13 cells, the final level of endogenous IAA is an equilibrium between increased synthesis of IAA and a massive release into the medium of the ester- and free-forms of IAA. Both cell lines show disturbances in embryogenesis: line 4w77 forms globular embryos that only mature into aberrant forms having multiple axes, whereas line 4w13 has completely lost its morphogenic capacity.