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)     

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.     

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

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.     

Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein.

cDNA clones representing genes that are expressed during leaf senescence in Brassica napus were identified by differential screening of a cDNA library made from RNA isolated from leaves at different stages of senescence. The expression of these genes at different stages of leaf development was examined by northern blot analysis, and several different patterns of expression were observed. One of the clones, LSC54, represented a gene that is expressed at high levels during leaf senescence. Analysis of this gene indicated strong expression in flowers as well as in senescing leaves. DNA sequence analysis of the LSC54 cDNA indicated a similarity between the deduced amino acid sequence and several metallothionein-like proteins previously identified in plants.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Natural Senescence of Pea Leaves (An Activated Oxygen-Mediated Function for Peroxisomes)

We studied the activated oxygen metabolism of peroxisomes in naturally and dark-induced senescent leaves of pea (Pisum sativum L.). Peroxisomes were purified from three different types of senescent leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. The activities of the O2-- and H2O2-producing enzymes were enhanced by natural senescence. Senescence also produced an increase in the generation of active oxygen species (O2- and H2O2) in leaf peroxisomes and in the activities of two glyoxylate-cycle marker enzymes. A new fraction of peroxisomes was detected at an advanced stage of dark-induced senescence. Electron microscopy revealed that this new peroxisomal fraction varied in size and electron density. During senescence, the constitutive Mn-superoxide dismutase (SOD) activity of peroxisomes increased and two new CuZn-SODs were induced, one of which cross-reacted with an antibody against glyoxysomal CuZn- SOD. This fact and the presence of glyoxylate-cycle enzymes support the idea that foliar senescence is associated with the transition of peroxisomes into glyoxysomes. Our results indicate that natural senescence causes the same changes in peroxisome-activated oxygen metabolism as dark-induced senescence, and reinforce the hypothesis of an effective role of peroxisomes and their activated oxygen metabolism in this stage of the life cycle.     

The Control of Autumn Senescence in European Aspen

The initiation, progression, and natural variation of autumn senescence in European aspen (Populus tremula) was investigated by monitoring chlorophyll degradation in (1) trees growing in natural stands and (2) cloned trees growing in a greenhouse under various light regimes. The main trigger for the initiation of autumn senescence in aspen is the shortening photoperiod, but there was a large degree of variation in the onset of senescence, both within local populations and among trees originating from different populations, where it correlated with the latitude of their respective origins. The variation for onset of senescence with a population was much larger than the variation of bud set. Once started, autumn senescence was accelerated by low temperature and longer nights, and clones that started to senescence late had a faster senescence. Bud set and autumn senescence appeared to be under the control of two independent critical photoperiods, but senescence could not be initiated until a certain time after bud set, suggesting that bud set and growth arrest are important for the trees to acquire competence to respond to the photoperiodic trigger to undergo autumn senescence. A timetable of events related to bud set and autumn senescence is presented.     

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.     

Senescence of Rice Leaves XVIII. Changes of Stomatal Aperture during Senescence

Changes of stomatal aperture during the course of developmentof rice leaves were directly observed with a scanning electronmicroscope. The stomata reached their maximal aperture sizeafter senescence began in seedling leaves and the flag leafof mature plants. The small stomatal aperture observed priorto senescence seems to be the normal size of stomata in riceleaves, and thus stomata closure does not seem to be the causeof leaf senescence in rice plants. The stomata retain theircapability of movement during senescence, suggesting that guardcells tend to live longer than mesophyll cells. ⁴Present address: Tobacco Taiwan, Republic of China ResearchInstitute, Taichung, Taiwan, Republic of China (Received March 12, 1987; Accepted September 30, 1987)     

Membrane Permeability--Regulation by Exogenous Sugars during Senescence of Oat Leaf in Light and Darkness

The effect of sugars (sucrose, glucose and fructose) on normalphysiological changes during senescence of foliar segments ofAvena sativa cv. Suregrain was studied. In general applicationof sugars raised tissue permeability both in the light and indarkness. This change was associated with increases in endogenoussugars, hydroperoxide content and lipoxygenase activity. Inthe light it was also associated with low catalase activity.Sugars did not influence superoxide dismutase activity. In thelight, sugars accelerated senescence, measured as decreasesin chlorophyll and increases in soluble amino acids. In darknesssugars delayed senescence. The effect of sugars in the lightseemed to result from an increase in photo-oxidations associatedwith the increase in permeability. The delaying effect on senescence,found in darkness, seemed to result from an increase in respiratoryactivity plus the lack of (or combined with the lack of) photo-oxidations. (Received March 18, 1985; Accepted June 3, 1986)     

Heritability and expected selection response for yield traits in blanched asparagus

Despite the continuous breeding that has been conducted with asparagus (Asparagus officinalis L.) since the beginning of the last century, there is little information on parameters for predicting direct and indirect selection response. Yield traits for blanched asparagus production were studied along a two-year period in a half-sib family population planted in Zavalla, Argentina. Half-sib family mean heritability values were low for total yield and marketable spear number (0.31 and 0.35), intermediate for marketable yield and total spear number (0.55 and 0.64), and relatively high for spear diameter and spear weight (0.75 and 0.74). An average increase in marketable yield of 15.9% is expected after each cycle of selection of the top 5% of the families. Total yield failed to express significant genetic correlations with any of the yield components; meanwhile marketable yield showed highly significant relations with market spear number (0.96) and spear weight (0.89). Indirect selection response over yield components (CRx) failed to be advantageous over direct selection (Rx), since the ratio CRx/Rx was always equal or below unity.