Identification and Characterization of an mRNA Encoding a Proline-Rich Protein that Rapidly Declines in Abundance in the Tips of Harvested Asparagus Spears

We previously isolated a cDNA clone, pTIP13, whose homologousmRNA rapidly declined in abundance in the tips of harvestedasparagus (Asparagus officinalis L.) spears [King and Davies(1992) Plant Physiol. 100: 1661]. In order to identify factorsregulating the postharvest deterioration of asparagus, we havenow sequenced the pTIP13 cDNA, derived the encoded amino acidsequence and determined the cellular location of pTIP13 mRNAby in situ hybridization. pTIP13 encodes a derived protein thatis rich in proline (22.3%), but also has a high content of lysine(15.2%) and threonine (14.1%). The proline residues are locatedin motifs at the amino-terminal region of the protein. The carboxyl-terminalregion of the derived protein has a high leucine content andshares >64% amino acid identity with derived proteins identifiedfrom cDNA clones to cell wall protein precursor mRNAs obtainedfrom soybean hypocotyls, alfalfa roots, and tomato fruit. GenomicSouthern analysis suggests that pTIP13 is encoded by a single-copygene in asparagus. pTIP13 mRNA was localized to specific celltypes in the young bracts of the asparagus spear tip. The resultsprovide new information on the complexity of tissue responsesin the tips of asparagus spears following harvest. (Received February 5, 1996; Accepted May 16, 1996)     

Physiological changes in asparagus spear tips after harvest

To extend our understanding of the physiology of asparagus after harvest, changes in respiration rate, protein and amino acid complement, and ultrastructure of tip sections (0–30 mm) of asparagus spears ( Asparagus officinalis L. cv. Limbras 10) were investigated. Spears had been stored for up to 4 days in the dark at 20°C. Respiration rate (carbon dioxide efflux) declined rapidly after harvest before stabilizing at 12 h at ca 50% of the rate at harvest. Protein, amino acid, and ammonium content of tip sections of 180 mm spears (intact tip sections) during storage, and comparable sections; excised from spears at harvest and subsequently stored (excised tip sections), were compared. Total protein content of intact and excised tip sections increased ca 10% 6–12 h after harvest, and then declined to ca 85% of harvest levels at 48 h. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed the net loss of specific proteins at 48 h. Free amino acid content of excised tip sections declined to ca 75% of harvest levels 12 h after harvest, and then increased to 150% of harvest levels by 48 h. Glutamine levels declined rapidly after harvest, and asparagine content increased ca 200% at 24 h. Similar trends in free amino acid content were found in sections of intact tips. Ammonia (ammonium ions) accumulated to ca 0.3% dry weight at 48 h in both intact and excised tip sections. Ultrastructural studies revealed that tonoplast breakdown commenced 48–96 h after harvest. Results are discussed in relation to the sequence of physiological events following harvest and the timing of mechanisms responsible for their initiation.     

Long-term effects of asparagus virus 2 infection on growth and productivity in asparagus

An asparagus field trial was established with clonal plants to determine the long-term effects of asparagus virus 2 (AV2) infection on spear production. Yield data, analysed by ANOVA, showed that AV2 infection caused significant (P < 0.05) decreases in spear yield which became more pronounced as the trial progressed. Mean marketable spear yields were reduced by 14%, 28%, 20%, 48% and 57% and reject spear yields were increased by 93%, 105%, 207%, 352% and 167%, during harvest years 1–5 respectively. Marketable spear yields from AV2–free plants increased annually to yr 5, but for AV2–infected plants, yields increased to yr 3 and decreased annually thereafter. Spears from AV2–infected plants were thinner than those from AV2–free plants, resulting in more reject thin spears by 109%, 88%, 220%, 499% and 216% during harvest yr 1–5, respectively. Further, data collected in yr 4 and 5 showed that AV2 infection had caused a 31 % reduction in mean spear diameter and reductions of 27% and 22% respectively, in diameter and height of fern stalks. Clearly, plants with smaller fern stalks were less able to accumulate carbohydrate reserves and therefore produced fewer, smaller spears and fern stalks the following spring. This may result in annual cycles of diminishing productivity in which the size and number of spears and fern stalks decrease with each successive year. The type and timespan of symptoms caused by AV2 infection in this trial are similar to those reported for asparagus decline syndrome and therefore it is likely that AV2 infection is a factor contributing to asparagus decline.     

Three differentially expressed basic peroxidases from wound-lignifying Asparagus officinalis

The activity of ionically bound peroxidases from an asparagus spear increased from 5-24 h post-harvest. Isoelectric focusing showed that the post-harvest increase of the total peroxidase activity was due to the increase of several distinct isoperoxidases. Concomitantly, a decrease in the activity of two anionic peroxidases was observed. Peroxidases with pI 5.9, 6.4 and 9.2 were detected only at 24 h post-harvest, whereas four peroxidases, with pI 8.7, 8.1, 7.4, and 6.7, detected throughout the time-course, increased in their activity. Histochemical staining demonstrated that lignin and peroxidase activity were located in the vascular bundles throughout the period of measurement. Lignin was detected in the cell walls of the protoxylem in the vascular bundles of the asparagus stem. A cDNA library of mRNA isolated from asparagus spears 24 h post-harvest was screened for peroxidases using homologous and heterologous probes. Three clones were isolated and the corresponding mature asparagus peroxidases displayed 70%, 76% and 81% amino acid sequence identity to each other. These new asparagus peroxidases are typical class III plant peroxidases in terms of conserved regions with a calculated pI >9.2, which is consistent with most basic peroxidases. One of the genes was shown to be a constitutively expressed single-copy gene, whereas the others showed an increased expression at post-harvest. The highest similarity in the amino acid sequence (71-77%) was found in peroxidases from roots of winter grown turnip TP7, to Arabidopsis AtP49, to an EST sequence from cotton fibres and to TMV-infected tobacco.     

Isoforms of glutamine synthetase in asparagus spears: the cytosolic enzyme increases after harvest

Two distinct forms of glutamine synthetase (GS) have been identified in the spear tip tissues of harvested asparagus (Asparagus officinalis L. cv. Limbras 10). The GS activities were separated by anion exchange chromatography. They have distinct kinetic properties and contain polypeptides of different sizes, and the abundances of the GS isoforms change differently after harvest. Plastid GS has a 44 kD polypeptide, and during the post-harvest period the abundance of this polypeptide declined dramatically. After 5 d, the activity of plastid GS had declined to just 20% of that at harvest. Cytosolic GS has a 40 kD polypeptide and is the major constituent of the GS activity present at harvest (73% of total). After harvest, cytosolic GS activity declined by half and then, at 3 or 4 d after harvest, rose to 80% of the cytosolic GS activity present at harvest. The nitrogen metabolism of asparagus spears is significantly altered as the tissues deteriorate rapidly after harvest. We demonstrate that cytosolic GS activity increases during the post-harvest period and is likely to be a critical feature of the physiology of the tip of a harvested asparagus spear.     

Novel jasmonate amino acid conjugates in Asparagus officinalis during harvest-induced and natural foliar senescence

Five jasmonates, including novel tryptophan conjugates of jasmonic acid and dihydrojasmonic acid, were identified in extracts from spears of Asparagus officinalis L. by electrospray tandem mass spectrometry. Spears were harvested and were held dry or with bases immersed in water. The concentrations of jasmonic acid, dihydrojasmonic acid, their tryptophan conjugates, cucurbic acid and methyl jasmonate, were measured by ELISA in spears in the 10 d following harvest. A transient increase that occurred in all spear tips immediately following harvest in the concentration of jasmonates can be attributed to a wounding response. A second increase in the concentration of jasmonates occurred from 7 d after harvest but only in dry-treated spear tips indicating that jasmonates may have accumulated in response to water stress. Jasmonate levels were also monitored during natural foliar senescence. Increased levels of jasmonates occurred after the onset of senescence, implicating them as a consequence rather than a cause of senescence.     

Activities of enzymes involved in lignification during the postharvest storage of etiolated asparagus spears

The content of lignin and the activities of 5 enzymes involved in lignification were monitored along the length of etiolated spears of asparagus ( Asparagus officinalis L., INRA Fl male hybrid n°156) stored for 22 h with their base in air (control), water or water containing the ethylene antagonist, silver thiosulfate (STS). At the time of harvest the lignin content increased basipetally, as did the activity of all the enzymes studied, viz., phenylalanine ammonia lyase (PAL; EC 4.3.1.5), hydroxycinnamate: CoA ligase (HCoAL; EC 6.2.1.12), cinnamoyl-CoA reductase (CCR: EC 1.2.1.44), cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) and syringaldazine oxidase (SyrOx. a peroxidase [POD; EC 1.11.1.7] with syringaldazine as substrate). Neither the lignin content nor the activity of any enzyme changed in the spear apex during storage, regardless of treatment. In the spear base. all enzyme activities decrased during the first 2 to 4 h in every storage treatment. Subsequently. PAL and HCoAL activities remained constant. whereas the activities of CAD and SyrOx gradually increased. Lignification in the spear base was not affected by storage in air. However, storage in water increased lignin formation and SyrOx activity, whereas treatment with STS prevented both of these increases. The results indicate that postharvest lignification in etiolated asparagus spears is caused primarily by enhanced SyrOx activity, and that ethylene is involved in the control of this activity.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

Spargelstangenuntersuchungen zur Haupterntezeit auf Infektionen mitFusarium spp. und Kontaminationen mit Fumonisin B1

Asparagus spears collected from a total of six commercial plantings in Austria during the main harvest periods in May and June of 2003 and 2004 were examined for endophytic colonization byFusarium spp., particularlyF. proliferatum. Potentially toxigenic fungi such asF. proliferatum were isolated and identified by morphological characteristics using light microscopy. Fumonisin B₁ inF. proliferatum-infected asparagus spears was detected with IAS-HPLC-FLD or HPLC-MS/MS. The identity of endophytic fungi colonizing of a total of 816 individual spears was determined. The incidence of infection byF. proliferatum and otherFusarium spp. was highly dependent on location and sampling date. The dominantFusarium species among the endophytic microflora wasF. oxysporum. Other frequently isolated species includedF. proliferatum, F. sambucinum, F. culmorum, F. avenaceum andF. equiseti. The incidence ofF. proliferatum-infected asparagus spears was less than 10% at four of the six sampling locations. At the two remaining locations, 20–47% of the spears examined were infected withF. proliferatum. Further exploration of FB₁ generation in asparagus is required because the low levels of FB₁ (10–50 (μg/kg) detected in harvested spears in 2003 and 2004 cannot be explained by the results of this study.

     

Bovine elastin cDNA clones: Evidence for the occurrence of a new elastin-related protein in fetal calf ligamentum nuchae

Poly (A+) mRNA was isolated from fetal calf ligamenturn nuchae and used for the construction of cDNA libraries. A fraction highly enriched in elastin mRNA was used to prepare the cDNA probes for screening the libraries. A 2 kb clone, pREl, gave the most positive signal in colony hybridization. It hybridized to a mRNA of the same size as reported for elastin mRNAs from chick and sheep. Hybrid-arrested translation showed that translation of mRNAs for proteins other than elastin doublet was not inhibited by pREI. Southern blot analysis showed that pREl has sequence homology with pVE6 and pVE10, which were tentatively identified as elastin-related cDNA clones representing two distinct mRNAs. DNA sequence data from the 5 end of pREl show that the translated amino acid sequence is not typical of known elastin sequences but contains some elastin-like sequences. All of this evidence strongly suggests the occurrence in fetal calf nuchal ligament of a mRNA which codes for a previously unknown elastin-related protein.     

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding

Two tomato cDNA libraries were synthesized from poly(A)⁺ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-(Tyr)₃-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-Thr-(Tyr)_1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)_2–6-Tyr-Pro and(Gly)_2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)_2–5-Arg repeats.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Cloning of two gibberellin-regulated cDNAs from Arabidopsis thaliana by subtractive hybridization: expression of the tonoplast water channel, γ-TIP,is increased by GA3

The Arabidopsis ga1 mutant has very low levels of endogenous, active gibberellins and thus has an extreme dwarf phenotype; application of GA₃ induces stem elongation and flower development. To test the hypothesis that GA action in this system involves changes in gene expression, we have cloned mRNAs whose abundance changes following GA application. A subtraction cloning scheme for the isolation of differentially regulated cDNAs was established, involving hybridization of single-stranded cDNA to biotinylated mRNA. cDNA populations enriched up to 150-fold in GA-regulated sequences were produced and cDNA libraries generated. Screening of these libraries has isolated two clones that identify mRNAs of ca. 1100 and 750 bases whose abundance is markedly increased 24 h after GA application. One of these clones encodes the vegetative form of the Arabidopsis tonoplast intrinsic protein (-TIP), a water channel protein, the expression of which has recently been shown to be correlated with regions of cell expansion. The second clone is expressed only in the inflorescence and encodes a proline- and glycine-rich protein that may be a cell wall component.     

Isolation and characterization of cDNA clones for castration-induced mRNAs in the rat ventral prostate.

To identify gene products involved in castration-induced involution of the rat ventral prostate, we constructed a subtraction cDNA library of the ventral prostate from rats castrated for 48 h. The library was screened with subtracted cDNA probes enriched for sequences with a low copy number expressed in intact or castrated rats. As a result of differential screening, 48 cDNA clones representing 10 different induced mRNAs were isolated. The time course of these mRNA inductions after castration was examined. Within the first 24 h after castration, the level of mRNAs for these cDNA clones was significantly increased and it reached its peak by 48-72 h after castration. Although mRNAs for these cDNA clones were expressed in various tissues from intact rats, an increase in mRNA as a response to castration was observed only in the ventral prostate. Partial sequence analyses of the 10 cDNA clones indicate that three cDNA clones represent rat glutathione S-transferase Yb-1, Yb-2 and Yb-3 subunit mRNA sequences, but for others respective homologues could not be found in a search of the GenBank database (release 67).     

Identification and characteristics of mRNA's showing increased levels during DNA replication

A library of double-stranded cDNA was prepared using poly (A) + RNA from regenerating rat liver 20 h after partial hepatectomy. Differential screening of 350 recombinant clones with cDNA-G0 and cDNA-S identified eleven cDNA clones (pRL), the sequences of which were preferentially expressed during the DNA replication period. Levels of mRNAs complementary to these clones were 2--10-fold higher in the S-period, than in G0. Using plasmid cDNAs to different mRNA, pRL we have investigated the changes in the levels of mRNA pRL during liver regeneration. The level of mRNA mRL2 and pRL79 was increased just before DNA replication. mRNA pRL35 accumulates after partial hepatectomy with the maximum at 6 h. The augment of two other mRNA concentrations was expressed to a lesser extent. Northern-blot analysis allowed to determine the individual dual mRNAs corresponding to each of the three clones with their sizes ranging from about 1650 to 3900 bases. Three mRNAs (pRL35, 67 and 79) were shown (by hybrid-selected translation) to code for proteins of about 100, 140 and 120 kDa, respectively.