Anthony D. Stead

A molecular and structural characterization of senescing Arabidopsis siliques and comparison of transcriptional profiles with senescing petals and leaves.

Senescence of plant organs is a genetically controlled process that regulates cell death to facilitate nutrient recovery and recycling, and frequently precedes, or is concomitant with, ripening of reproductive structures. In Arabidopsis thaliana, the seeds are contained within a silique, which is itself a photosynthetic organ in the early stages of development and undergoes a programme of senescence prior to dehiscence. A transcriptional analysis of the silique wall was undertaken to identify changes in gene expression during senescence and to correlate these events with ultrastructural changes. The study revealed that the most highly up-regulated genes in senescing silique wall tissues encoded seed storage proteins, and the significance of this finding is discussed. Global transcription profiles of senescing siliques were compared with those from senescing Arabidopsis leaf or petal tissues using microarray datasets and metabolic pathway analysis software (MapMan). In all three tissues, members of NAC and WRKY transcription factor families were up-regulated, but components of the shikimate and cell-wall biosynthetic pathways were down-regulated during senescence. Expression of genes encoding ethylene biosynthesis and action showed more similarity between senescing siliques and petals than between senescing siliques and leaves. Genes involved in autophagy were highly expressed in the late stages of death of all plant tissues studied, but not always during the preceding remobilization phase of senescence. Analyses showed that, during senescence, silique wall tissues exhibited more transcriptional features in common with petals than with leaves. The shared and distinct regulatory events associated with senescence in the three organs are evaluated and discussed.

A Comparison of Leaf and Petal Senescence in Wallflower Reveals Common and Distinct Patterns of Gene Expression and Physiology

Petals and leaves share common evolutionary origins but perform very different functions. However, few studies have compared leaf and petal senescence within the same species. Wallflower (Erysimum linifolium), an ornamental species closely related to Arabidopsis (Arabidopsis thaliana), provide a good species in which to study these processes. Physiological parameters were used to define stages of development and senescence in leaves and petals and to align these stages in the two organs. Treatment with silver thiosulfate confirmed that petal senescence in wallflower is ethylene dependent, and treatment with exogenous cytokinin and 6-methyl purine, an inhibitor of cytokinin oxidase, suggests a role for cytokinins in this process. Subtractive libraries were created, enriched for wallflower genes whose expression is up-regulated during leaf or petal senescence, and used to create a microarray, together with 91 senescence-related Arabidopsis probes. Several microarray hybridization classes were observed demonstrating similarities and differences in gene expression profiles of these two organs. Putative functions were ascribed to 170 sequenced DNA fragments from the libraries. Notable similarities between leaf and petal senescence include a large proportion of remobilization-related genes, such as the cysteine protease gene SENESCENCE-ASSOCIATED GENE12 that was up-regulated in both tissues with age. Interesting differences included the up-regulation of chitinase and glutathione S-transferase genes in senescing petals while their expression remained constant or fell with age in leaves. Semiquantitative reverse transcription-polymerase chain reaction of selected genes from the suppression subtractive hybridization libraries revealed more complex patterns of expression compared with the array data.

HAWAIIAN SKIRT: An F-Box Gene That Regulates Organ Fusion and Growth in Arabidopsis

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, ProPGAZAT:β-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using ProHWS:β-glucuronidase or ProHWS:green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro35S:HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.

Increasing flower longevity in Alstroemeria

The vase-life of Alstroemeria (cv. Rebecca) flowers is terminated when the tepals abscise. Abscission was accelerated by both chloroethylphosphonic acid (CEPA) and 1-aminocyclopropane-1-carboxylic acid (ACC). Petals abscised 24 h earlier compared with controls, when isolated cymes were placed in 340 nM CEPA, and earlier still when higher concentrations were used. This suggests that flowers of this Alstroemeria cultivar are very ethylene sensitive. Treatment with silver thiosulphate (STS) overcame the effects of exposure to CEPA and delayed perianth abscission of untreated isolated flowers by 3-4 days. The inclusion of 1% sucrose in the vase solution also extended longevity but not by as much as STS treatment; combined STS and sucrose treatments did not increase longevity beyond that of either treatment alone. However, removal of the young buds from the axil of the first flower was the most effective treatment to extend vase-life and encouraged the growth and development of the remaining flower. Flowers on cut inflorescences from which young axillary buds were trimmed more than doubled in fresh weight 6 days after flower opening compared with an increase of only 70-80% in those untreated or treated with STS and/or sucrose. Growth was less in isolated cymes but followed a similar pattern. The effect of STS and/or sucrose treatment was synergistic with the trimming treatment and thus the vase-life of trimmed, STS and sucrose-treated flowers was over 7 days longer than that for untreated controls.

Proteomic analysis of pollination-induced corolla senescence in petunia

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petunia×hybrida ‘Mitchell Diploid’ corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

Assay of the polyamine spermine by a monoclonal antibody-based ELISA

Spermine-specific monoclonal antibodies were prepared by immunising mice with protein-spermine conjugates. A resulting monoclonal antibody, IAG-1, exhibited both high affinity for spermine (binding constant 5.5 x 10(7) M-1) and high specificity, cross-reacting only weakly with spermidine. Using this antibody a competitive ELISA was developed with a detection limit of 1 pmol. The assay has been used to quantify spermine content of plant tissues, without derivatization, and producing within 6 h of collection, values for the spermine content which are similar to published data obtained by HPLC.

The Use of Soft X Rays to Study the Ultrastructure of Living Biological Material

Imaging biological specimens with soft x rays offers several potential benefits over electron microscopy, and these are briefly reviewed. The disadvantages, most notably radiation-induced structural changes, have been investigated and images of irradiated algal cells (Chlorella) are presented. In soft x-ray contact microscopy the image is recorded rapidly to avoid both natural and radiation-induced movement and this technique has been used to study the ultrastructural effects of electron microscopy fixatives. In the epidermal hairs of tomato plants there are numerous strands of cytoplasm which, by light microscopy, appear to traverse the vacuole but are rarely seen by electron microscopy. However, by soft x-ray contact microscopy these strands and the organelles within them can be successfully imaged. Moreover, examination by soft x-ray contact microscopy of the cytoplasm in a fixed material shows that these strands are not present in chemically fixed material. This paper also reports the use of soft x-ray contact microscopy to examine the abscission cells found within the protonema of a moss (Bryum tenuisetum) and compares the images to those obtained by light and electron microscopy.

The formation of aplastidic abscission (tmema) cells and protonemal disruption in the mossBryum tenuisetum Limpr. is associated with transverse arrays of microtubules and microfilaments

SummaryWhen grown on nutrient agar, protonemata ofBryum tenuisetum produce aerial filaments containing several abscission or tmema cells (TC). Basipetal migration of the nucleus and some of the chloroplasts signals the onset of TC formation. This is followed by the creation of a plastid-free zone at the base of the mother cell. The ensuing cytokinesis produces a very short aplastidic TC. This expands without the deposition of new wall material. Eventually the wall ruptures around the equator thus disrupting the protonemal filament. The site of wall breakdown is marked by a narrow band of cortical cytoplasm containing colocalized circumferential rings of actin filaments and microtubules. A transverse band of microtubules appears at the extreme basal end of the tmema mother cell. This band, which is not colocalized with actin filaments, migrates distally over the surface of the nucleus. Intimate spatial and developmental correlations suggest that this transverse array of the microtubules has a key role in excluding plastids from the TC. It is therefore considered not to be homologous with a preprophase band.

Petunia Flower Senescence

Senescence represents the last stage of floral development and is an active process that requires gene transcription and protein translation. A genetically controlled senescence program allows for the ordered degradation of organelles and macromolecules and the remobilization of essential nutrients from the petals. Petunia provides an excellent model system for studies of flower senescence because the plants flower profusely and have large floral organs amenable to molecular and biochemical analysis. While Petunia flowers have a finite lifespan that is under tight developmental control, petal senescence can be accelerated and synchronized by means of exogenous ethylene or by pollination. Petal senescence in Petunia is accompanied by decreased nucleic acid and protein content, DNA and nuclear fragmentation, and structural and compositional changes in the plasma membrane. These changes are correlated with increased mRNA abundance and enzyme activity of proteases, nucleases, and phospholipases. Major macronutrient levels in Petunia petals (collectively called the corolla) also decrease during senescence. These studies support cellular degradation and remobilization as the central functions of petal senescence. Ethylene is clearly involved in modulating the process, but the transcription factors and other components of the senescence signal transduction pathway(s) remain to be elucidated. Further studies focusing on early transcriptome changes during petal senescence will help to identify these early regulators, and subsequent studies of protein changes and the post-translational modification of senescence-related proteins will further our understanding of the pathways executing the senescence program.

Atomic force microscopy employed as the final imaging stage for soft x-ray contact microscopy

Soft x-ray contact microscopy (SXCM) enables a high resolution image of a living biological specimen to be recorded in an x-ray sensitive photoresist at unity magnification. Until recently scanning electron microscopes (SEM) have been employed to obtain the final magnified image. Although this has been successful in producing many high resolution images, this method of viewing the resist has several disadvantages. Firstly, a metallic coating has to be applied to the resist surface to provide electrical conductivity, rendering further development of the resist impossible. Also, electron beam damage to the resist surface can occur, in addition to poor resolution and image quality. Atomic force microscopy (AFM) allows uncoated resists to be imaged at a superior resolution, without damage to the surface. The use of AFM is seen as a major advancement in SXCM. The advantages and disadvantages of the two technologies are discussed, with illustrations from recent studies of a wide variety of hydrated biological specimens imaged using SXCM.