Systemic signalling of environmental cues in Arabidopsis leaves.

Light intensity and atmospheric CO2 partial pressure are two environmental signals known to regulate stomatal numbers. It has previously been shown that if a mature Arabidopsis leaf is supplied with either elevated CO2 (750 ppm instead of ambient at 370 ppm) or reduced light levels (50 micromol m-2 s-1 instead of 250 micromol m-2 s-1), the young, developing leaves that are not receiving the treatment grow with a stomatal density as if they were exposed to the treatment. But the signal(s) that it is believed is generated in the mature leaves and transmitted to developing leaves are largely unknown. Photosynthetic rates of treated, mature Arabidopsis leaves increased in elevated CO2 and decreased when shaded, as would be expected. Similarly, the levels of sugars (glucose, fructose, and sucrose) in the treated mature leaves increased in elevated CO2 and decreased with shade treatment. The levels of sugar in developing leaves were also measured and it was found that they mirrored this result even though they were not receiving the shade or elevated CO2 treatment. To investigate the effect of these treatments on global gene expression patterns, transcriptomics analysis was carried out using Affymetrix, 22K, and ATH1 arrays. Total RNA was extracted from the developing leaves after the mature leaves had received either the ambient control treatment, the elevated CO2 treatment, or the shade treatment, or both elevated CO2 and shade treatments for 2, 4, 12, 24, 48, or 96 h. The experiment was replicated four times. Two other experiments were also conducted, one to compare and contrast gene expression in response to plants grown at elevated CO2 and the other to look at the effect of these treatments on the mature leaf. The data were analysed and 915 genes from the untreated, signalled leaves were identified as having expression levels affected by the shade treatment. These genes were then compared with those whose transcript abundance was affected by the shade treatment in the mature treated leaves (1181 genes) and with 220 putative 'stomatal signalling' genes previously identified from studies of the yoda mutant. The results of these experiments and how they relate to environmental signalling are discussed, as well as possible mechanisms for systemic signalling.

Suppression of the cysteine protease, aleurain, delays floret senescence in Brassica oleracea.

An aleurain-like protein, BoCP5, is up-regulated during harvest-induced senescence in broccoli floret and leaf tissue. BoCP5 is most closely related to an Arabidopsis protein (91%, AAF43041) and has 71% identity to barley aleurain (P05167). The mRNA for this gene accumulates within 6 h after harvest in broccoli florets, and its expression is reduced in tissue that has been held in senescence-delaying treatments (e.g. water, sucrose feeding, controlled atmosphere). The gene is also expressed in leaves during aging-related and harvest-induced senescence. Analysis of protein bands that cross-react with antibodies raised to the bacterial BoCP5 fusion protein, revealed prominent immunoreactive bands at ca. 26, 28, 31, and 38 kD in floret tissue. The 31 kD band was absent in protein extracts from leaf tissue. Agrobacterium-mediated transformation was used to produce transgenic broccoli plants with down-regulated BoCP5. A reduction in the postharvest expression of BoCP5 in floret tissue was achieved for four transgenic lines in the current study. In three of these lines postharvest floret senescence (yellowing) was delayed, and florets contained significantly greater chlorophyll levels during postharvest storage at 20 degrees C than wild-type plants. Line 4 showed the greatest down-regulation of BoCP5, and in this line postharvest protease activity remained at pre-harvest levels, and the yield of soluble proteins extracted from florets after harvest was significantly greater than that of wild-type tissue.

Characterization of a DNA-binding protein that interacts with 5' flanking regions of two fruit-ripening genes.

The E4/E8 binding protein (E4/E8BP) interacts with sequences in the 5' flanking regions of two genes, E4 and E8, that are coordinately regulated by ethylene during tomato fruit ripening. The DNA-binding activity of this protein increases during fruit ripening, and it may play a role in regulation of these genes. To begin to understand the function of this protein, a cDNA has been isolated that encodes a protein, E4/E8BP-1, with DNA-binding specificity similar to that of E4/E8BP. This DNA-binding protein is closely related to a DNA binding protein from tobacco, 3AF1, that interacts with the promoter of the pea rbcS-3A gene. A repeated domain was identified within the predicted 3AF1 amino acid sequence, which includes a series of histidines and cysteines, suggestive of zinc binding, and this repeat is conserved in E4/E8BP-1. Interaction of both E4/E8BP-1 and nuclear extracts from ripening fruit with the E8 recognition sequence is sensitive to 1,10-phenanthroline, indicating that a metal is required for binding of both the native and recombinant proteins. The mRNA for E4/E8BP-1 is moderately abundant in fruit, and increases slightly during fruit ripening, consistent with a role in fruit ripening. A truncated version of E4/E8BP-1 was able to transactivate the E4 promoter in transient assay, demonstrating that this DNA-binding protein can interact with the E4 promoter in vivo to enhance gene transcription.

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 cysteine-rich (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.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and accumulation pattern of an mRNA encoding an abscission-related beta-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 beta-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 beta-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.

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, 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.

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.