The citrus fruit proteome: insights into citrus fruit metabolism.

Fruit development and ripening are key processes in the production of the phytonutrients that are essential for a balanced diet and for disease prevention. The pathways involved in these processes are unique to plants and vary between species. Climacteric fruit ripening, especially in tomato, has been extensively studied; yet, ripening of non-climacteric fruit is poorly understood. Although the different species share common pathways; developmental programs, physiological, anatomical, biochemical composition and structural differences must contribute to the operation of unique pathways, genes and proteins. Citrus has a non-climacteric fruit ripening behavior and has a unique anatomical fruit structure. For the last few years a citrus genome-wide ESTs project has been initiated and consists of 222,911 clones corresponding to 19,854 contigs and 37,138 singletons. Taking advantage of the citrus database we analyzed the citrus proteome. Using LC-MS/MS we analyzed soluble and enriched membrane fractions of mature citrus fruit to identify the proteome of fruit juice cells. We have identified ca. 1,400 proteins from these fractions by searching NCBI-nr (green plants) and citrus ESTs databases, classified these proteins according to their putative function and assigned function according to known biosynthetic pathways.

Homeodomain leucine zipper proteins bind to the phosphate response domain of the soybean VspB tripartite promoter.

The soybean (Glycine max L. Merr. cv Williams 82) genes VspA and VspB encode vacuolar glycoprotein acid phosphatases that serve as vegetative storage proteins during seed fill and early stages of seedling growth. VspB expression is activated by jasmonates (JAs) and sugars and down-regulated by phosphate and auxin. Previous promoter studies demonstrated that VspB promoter sequences between -585 and -535 mediated responses to JA, and sequences between -535 and -401 mediated responses to sugars, phosphate, and auxin. In this study, the response domains were further delineated using transient expression of VspB promoter-beta-glucuronidase constructs in tobacco protoplasts. Sequences between -536 and -484 were identified as important for phosphate responses, whereas the region from -486 to -427 mediated sugar responses. Gel-shift and deoxyribonuclease-I footprinting assays revealed four DNA-binding sites between -611 and -451 of the soybean VspB promoter: one in the JA response domain, two in the phosphate response domain, and one binding site in the sugar response domain. The sequence CATTAATTAG present in the phosphate response domain binds soybean homeodomain leucine zipper proteins, suggesting a role for these transcription factors in phosphate-modulated gene expression.

NADP(+)-isocitrate dehydrogenase gene expression and isozyme activity during citrus fruit development.

The accumulation of citric acid and its decline toward fruit maturation is typical of citrus fruit. We studied NADP(+)-isocitrate dehydrogenase (NADP-IDH), an enzyme involved in citrate metabolism. A cDNA encoding the enzyme was cloned from lemon (Citrus limon) juice sac cells, and is the first-reported NADP-IDH from fruits. Sequence comparisons and phylogenetic analysis indicate that it most probably belongs to a monophyletic clade of plant cytosolic enzymes. The mRNA level in the juice sac cells was induced during lemon fruit growth, and increased by about 15-fold to a peak as the fruit neared maturation. Spectrophotometric assay of the NADP-IDH activity in the pulp during fruit development showed that in young fruit, most of the activity was associated with the mitochondrial preparation and that, as the fruit grew, the activity shifted to the soluble fraction. The two activities could also be distinguished by isozyme gel electrophoresis: while one isozyme was detected in the mitochondrial preparation of young fruit and declined later, the other was induced in the soluble fraction of older fruit and increased as the fruit grew. The increasing activity of NADP-IDH in the soluble fraction throughout fruit development correlated well with the increase in gene expression, which suggests that the soluble activity is regulated by the expression of the cytosolic NADP-IDH gene. The possible role of this form of the enzyme in citric acid catabolism in the pulp is discussed.

Fermentative metabolism in grape berries: isolation and characterization of pyruvate decarboxylase cDNA and analysis of its expression throughout berry development.

The involvement of pyruvate decarboxylase (PDC) in the control of alcohol production during ripening of fruit tissues under aerobic conditions has been only partially studied. Enzymological studies showed a significant increase in PDC activity during the ripening of oranges and pears, concurrently with the induction of ethanol production. In tomato, on the other hand, the induction of ethanol production and ADH gene expression after the onset of ripening was not accompanied by induction of PDC activity. The isolation of PDC cDNA from fruits has not yet been reported, nor has its expression pattern during fruit development. We report here the isolation of a cDNA clone encoding for a grape PDC and the characterization of its expression throughout berry development. The pattern of PDC gene expression throughout berry development, combined with earlier findings on constitutive PDC activity in the berry, may suggest that PDC is not the limiting factor for the production of ethanol in the berry, which is induced only after the onset of berry ripening. Alternatively, the induction of ADH gene expression, which occurs only after the onset of ripening in both tomatoes and grape berries, may serve as a regulator of ethanol production in response to a ripening-related cue.

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation.

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA3 pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate.

The soybean vegetative storage proteins, VSP alpha and VSP beta, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp alpha and Vsp beta, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.

Phosphate Modulates Transcription of Soybean VspB and Other Sugar-Inducible Genes.

The soybean vegetative storage protein genes VspA and VspB encode vacuolar glycoprotein acid phosphatases. Transcription of the Vsp is synergistically activated by jasmonic acid or methyl jasmonate (MeJA) and soluble sugars. The action of these modulators is mediated by two different DNA domains in the VspB promoter. In this study, we present new data regarding VspB regulation by sucrose and inorganic phosphate, which suggest a common mechanism of transcriptional control for Vsp and other sugar-inducible genes. We found that the sugar-mediated activation of VspB expression was inhibited by phosphate. Deletion analysis and transient assays in tobacco protoplasts identified a 130-bp DNA domain in the VspB promoter that mediates both sucrose induction and phosphate inhibition. Transcription mediated by this DNA domain was induced by phosphate elimination from the protoplast incubation medium, even in the absence of sucrose. The effect of sucrose and phosphate on VspB expression was studied in vivo in several ways. Depletion of phosphate from soybean cell cultures by the addition of mannose stimulated VspB expression, even in the absence of sucrose or MeJA. In illuminated soybean leaves treated with MeJA, inhibition of photosynthetic electron transport by DCMU decreased VspB expression. In contrast, VspB expression in soybean leaves stimulated by phosphate depletion was not influenced by DCMU. Moreover, sucrose-stimulated expression of the sugar-responsive genes lipoxygenase A and chalcone synthase of soybean and proteinase inhibitor II and class I patatin of potato was inhibited by phosphate. Like VspB, these genes were stimulated by phosphate depletion in the absence of exogenous sucrose. We propose that sugar-responsive genes are activated, in part, by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. These data may help explain recruitment of the Vsp, which encode acid phosphatases, as vegetative storage proteins.

Sucrose Modulation of Soybean Vsp Gene Expression Is Inhibited by Auxin.

We have shown that auxin represses soybean (Glycine max L.) vegetative storage protein gene (Vsp) expression in suspension-cultured cells and in leaves and petioles of excised trifoliates. The auxin analog naphthyleneacetic acid (NAA) at 10 [mu]M strongly inhibited methyl jasmonate-induced Vsp expression in soybean suspension-cultured cells. Both indole-3-acetic acid and NAA inhibited methyl jasmonate- and wound-induced expression of the Vsp and LoxA excised soybean trifoliate leaves and petioles. The less active auxin analog phenylacetic acid had less effect on methyl jasmonate- and wound-induced expression of these genes. Addition of cytokinin to alter the auxin:cytokinin ratio did not reverse auxin inhibition of Vsp expression. Transcription of [beta]-glucuronidase (Gus) modulated by a methyl jasmonate-responsive domain derived from the VspB promoter was minimally influenced by auxin. In contract, sucrose-induced expression of Gus mediated by a sucrose-responsive domain of the VspB promoter was strongly inhibited by NAA. We conclude that auxin inhibits Vsp mRNA accumulation, in part, by repressing sugar-mediated activation of Vsp expression.

A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina.

Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M(r) cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface.

Use of monoclonal antibodies in the purification of an inhibitor of virus replication by affinity chromatography.

Mouse monoclonal antibodies (MAbs) were prepared to an inhibitor of virus replication (IVR), released from protoplasts or leaf tissue of hypersensitive tobacco plants infected with tobacco mosaic virus. The MAbs were highly specific for IVR and reduced its antiviral activity. Using these MAbs in affinity chromatography enabled the recovery of purified IVR. SDS-PAGE of the immunoaffinity-purified IVR gave a single Mr 23K band. Immunoblots of IVR from extracts of protoplast or leaf tissue also revealed a single Mr 23K band which suggests that protoplast and tissue IVR are closely related.