Simon A. Coupe

Characterisation of an mRNA encoding a metallothionein-like protein that accumulates during ethylene-promoted abscission of Sambucus nigra L. leaflets

A cDNA encoding a metallothionein-like protein has been isolated from a cDNA library from the abscission zones of ethylene-treated Sambucus nigra leaflets. The precise function of this group of proteins in plants has yet to be confirmed but in animals there is convincing evidence that they bind heavy metals. Several of these proteins have recently been characterised from plants and it has been demonstrated that heavy metals have no stimulatory effect on their expression. In this paper we describe the isolation and characterisation of a metallothionein-like mRNA identified as a consequence of differentially screening a cDNA library for messages up-regulated during abscission. The accumulation of the mRNA occurred in the abscission zone tissue within 18 h of exposure to ethylene while, in contrast, no expression was detectable in adjacent non-abscission-zone tissue. The transcript size of the message was approximately 0.6 kb. Northern analysis revealed that the cDNA insert (JET12) did not hybridise to mRNA from either green or senescing leaflets but a signal was detectable with mRNA extracted from senescent tissue. The size of this hybridising transcript was approximately 0.5 kb. The predicted metallothionein-like protein encoded by JET12 was cysteinerich (18.4%) and had a molecular weight of approximately 7.5 kDa. Southern analysis of S. nigra genomic DNA showed that the mRNA was encoded by a small gene family. The protein exhibited greatest homology to other metallothioneins belonging to the Type 2 family including those from Mimulus (62%) and Arabidopsis (57%). This homology was greatest around the cysteine-rich amino and carboxy termini. The possible role of the protein encoded by JET12 during ethylene-promoted leaflet abscission is discussed.

Characterization and accumulation pattern of an mRNA encoding an abscission-related β-1,4-glucanase from leaflets of Sambucus nigra

A cDNA library was produced using mRNA extracted from ethylene-treated leaflet abscission zones of common elder (Sambucus nigra). Screening of the library with the insert from pBAC10, which encodes an abscission β-1,4-glucanase (cellulase) from bean (Phaseolus vulgaris), resulted in the isolation of a near-full-length cDNA which was designated JET 1. Northern analysis, using JET 1 as a probe, detected a transcript of 1.9 kb that accumulated prior to the first visible signs of cell separation. Accumulation of the JET 1 transcript is promoted by ethylene and primarily restricted to the tissue comprising the abscission zone. Sequence analysis of JET 1 indicates it is 1768 bp in length and shares significant homology at the amino acid level with β-1,4-glucanases from the leaf abscission zone of P. vulgaris (67%) and ripening avocado fruit (48%). The predicted peptide sequence of the S. nigra enzyme contains two potential glycosylation sites. Genomic Southern analysis of S. nigra DNA reveals that JET 1 may belong to a multi-gene family.

Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.)

Pod development in oilseed rape (Brassica napus) culminates in a process known as dehiscence (shatter) which can result in the loss of seed before the crop is harvested. In order to investigate the biochemical and the genetic basis controlling this process, a cDNA library was constructed from the dehiscence zone of developing pods. This resulted in the isolation of a cDNA clone (SAC51). The mRNA encoded by SAC51 had a transcript size of ca. 700 nucleotides and was found, by northern analysis, to accumulate preferentially in the dehiscence zone of the pod and in no other part of the plant analysed. The predicted polypeptide is rich in the amino acids proline (14.2%) and leucine (14.2%). The sequence of the polypeptide has more than 40% amino acid sequence identity with polypeptides isolated from carrot embryos, maize roots, soybean seeds and young tomato fruit. The function of these proteins is unknown. Genomic Southern analysis suggests that SAC51 is encoded by a single gene or small gene family. The role of the peptide in the development of pods of oilseed rape is discussed.

Characterization of a mRNA that accumulates during development of oilseed rape pods

Dehiscence of oilseed rape pods, commonly known as pod shatter, is a process of agronomic importance that results in seed loss causing yield reductions and carry-over of the crop into the following growing season. In an effort to understand the mechanisms underlying this developmental event, the changes in gene expression that accompany pod shatter have been examined with a view to understanding how the process is regulated. In order to achieve this, a cDNA library was constructed using mRNA extracted from the dehiscence zone of developing pods. Differential screening with non-dehiscence zone cDNA led to the isolation of a pod-specific clone, SAC25, with a transcript size of 1100 nucleotide encoding a predicted polypeptide of 34 kDa. The level of SAC25 mRNA accumulation increased during pod development. The sequence shows no significant homology to others within the databases but has two identifiable amino acid motifs, one is an adenine nucleotide binding site for NAD/FAD dehydrogenases and the other is a conserved feature of the ribitol dehydrogenase family. The amino acid sequence has four putative glycosylation sites and contains four cysteine residues. Genomic Southern analysis indicates that SAC25 may be encoded by a single gene or a small gene family. The function of this mRNA is unknown but possible roles in dehiscence and pod development are discussed.

Controlled atmospheres and sugar can delay malate synthase gene expression during asparagus senescence

A cDNA clone encoding malate synthase (MS; EC 4.1.3.2) was isolated from a 48-h postharvest asparagus (Asparagus officinalis L.) spear cDNA library using a MS clone from Brassica napus. The asparagus MS (AoMS1) cDNA hybridized to a 1.9-kb transcript that increased in abundance preferentially in spear-tip tissue during postharvest storage. The AoMS1 transcript also accumulated during natural foliar senescence of asparagus fern. The cDNA consists of 1960 nucleotides with an open reading frame of 1665 nucleotides or 555 amino acids, and encodes a deduced protein with a predicted Mr of 63 kDa and a pI of 8.1. The deduced amino acid sequence of AoMS1 showed high identity with the B. napus MS clone (77.2%) used to isolate it, and with MS from cucumber (77%). Genomic Southern analysis suggests that a single gene in asparagus encodes AoMS1. Controlled- atmosphere treatments aimed at reducing deterioration of harvested asparagus spears reduced the expression of AoMS1. The reduction was correlated with the reduced oxygen level, and reduced MS enzyme activity was also observed. Asparagus cell cultures were used to test the role of sugar status in regulating AoMS1 gene expression. In cultures without sucrose there was an accumulation of AoMS1 transcript that was absent in cultures containing sucrose.

Xyloglucan endotransglycosylase: a role after growth cessation in harvested asparagus

Little is known about the mode of xyloglucan endotransglycosylase (XET) activity in cell walls once the turgor, which drives expansion, is reduced. Such a situation exists when growing shoots are excised from the parent plant, and is the case for many commercially valuable vegetable crops, e.g. asparagus, Asparagus officinalis L. XET activity was present in all zones of rapidly growing, immature asparagus spears, but with highest levels at the spear base where elongation growth had ceased. Activity increased in all parts of the spear for up to 72 h after harvest. Two members of the XET-related gene family in asparagus (AoXET1 and AoXET2) were isolated and mRNA corresponding to these clones accumulated at low levels, particularly in the basal zone during spear growth. Transcript levels increased in all parts of the asparagus spear after harvest, but this increase did not coincide with the increase in XET activity. The harvest-related changes to xyloglucan molecular weight were restricted to slight, segment-specific, up- or down-shifts. However, this may hide strategic alterations to linkages leading to a more rigid wall without major changes in overall molecular weight. The initial postharvest surge in XET activity could be related to harvest stresses such as water deficit, but we propose that the later induction of AoXET1 and AoXET2 is linked to the development of lignified secondary cell walls.

Spatial and Temporal Expression of Abscission-Related Genes During Ethylene-Promoted Organ Shedding

During the course of development a spectrum of organs are shed from a plant [1]. Although the cellular and molecular mechanisms by which the abscission process is brought about have yet to be ascertained, the final event is the dissolution of the cell wall at the site where shedding takes place. A major difficulty encountered when studying abscission is that only a few rows of cells may undergo separation. In bean, there is convincing evidence that only one or two rows of cells may contribute to the loss of the primary leaf [2] while in tomato flowers there may be 5-10 rows of cells involved in the process [3]. In an attempt to examine the molecular changes which lead to abscission we have chosen to study the shedding of leaflets in Sambucus nigra [4,5,6]. This is a well characterised system and as the leaflet abscission zone is a composite of three sites where cell separation takes place, as many as 50 rows of cells may be involved in the process [7]. Moreover, leaflet abscission can be induced in approximately 24 hours by exposure to ethylene (10 μ1 1-1) and the use of explants leads to a close degree of synchronisation of the process [5].

Changes in gene expression during leaf abscission

Abscission of the leaflets of Sambucus nigra is stimulated by ethylene. Associated with cell separation is an increase in the activity of β 1,4 glucanase (cellulase) and polygalacturonase (PG) and this rise is restricted to the cells comprising the abscission zone. A cDNA library has been generated from mRNA extracted from separating abscission zone cells and screened using a heterologous s 1,4 glucanase probe from Phaseolus vulgaris. A number of positive cDNAs have been isolated from the S. nigra library and the largest clone designated pJET1 has a size of 1.7Kb. pJET1 has been sequenced and exhibits over 70% deduced amino acid homology with the protein encoded by the P. vulgaris cDNA. Using a differential screening strategy over 20 abscission-related cDNAs have been isolated from the library. None of these hybridizes to pJET1 and their role in abscission remains to be determined.