Dr Hilary Rogers - PhD
1) Plant Organ senescence
A) SAG21/AtLEA5 a gene at the interface between stress responses and senescence
SAG21 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, in collaboration with Prof Christine Foyer (Leeds) and Dr Frederica Theodoulou (Rothamsted) we characterised root and shoot development and response to biotic stress in SAG21 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass (Fig 1) (Mohd Salleh et al, 2012)
Fig 1: A: Aradopsis lines in which SAG21 is over-expressed (OEX) B: and down regulated by antisense (AS) showing effects on (A) growth and (B) senescence (Mohd Salleh et al, 2012)
Expression of SAG21 is induced by numerous abiotic stresses (Fig 2A) and in collaboration with Dr Luis Mur (Aberystwyth) we also investigated whether perturbation of SAG21 affected growth of pathogens. We found that growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21 expression, however growth of an avirulent P.syringae strain was unaffected (Fig. 2B). In collaboration with Dr John Runions (Oxford Brookes) we showed that a SAG21 -YFP fusion was localised to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling which in turn, impacts on root development and pathogen responses.
Fig 2:A: Response of SAG21 promoter GUS lines to a range of abiotic stresses B: Aniline blue staining of B.cinerea hyphae in SAG21 transgenic plants and WT 24 h and 48 h post-inoculation (Mohd Salleh et al, 2012)
B) Floral Senescence
Floral senescence in many species is largely controlled by the plant growth regulator ethylene. However, the senescence of many economically important species is not ethylene sensitive and therefore the techniques presently available are ineffectual at prolonging their storage or vase life. How floral senescence in these species is regulated remains an interesting biological question. In collaboration with Dr A Stead (Royal Holloway), Dr B Thomas, and Dr V Buchanan-Wollaston (University of Warwick HRI), we investigated the biochemical and molecular events occurring during floral senescence in an important UK cut flower crop Alstroemeria (Wagstaff et al. 2002a,b; 2003; Leverentz et al., 2003). This species shows ethylene independent floral senescence (Wagstaff et al, 2005) with flowers lasting up to 15 days under optimal conditions. Using microarrays we have shown that large numbers of transcripts are up-and down-regulated during senescence (Breeze et al. 2004).
Fig. 3 Stages of Alstroemeria flower development and senesence and effect of post-harvest treatments on vase life (Breeze et al, 2004, Wagstaff et al, 2010)
We were also interested in discovering what the overlap in gene expression is between developmental senescence, and premature senescence induced by environmental stress such as ambient dehydration and cold storage. This has implications both for understanding the regulation of senescence regulatory networks and has practical implications in the cut flower industry where flowers are often stored in suboptimal conditions during the transport chain. Microarray analysis revealed that there was significant sharing of gene expression between developmental senescence and an ambient dry stress treatment, whereas cold induced a distinct profile of transcripts (Wagstaff et al., 2010) (Fig. 3).
Fig 4: GC-MS trace showing a peak of myrcene produced by Alstroemeria cv. Sweet Laura flowers (Aros et al, 2012)
One of the transcripts whose expression changed during cold treatments is a terpene synthase-like gene and in collaboration with Dr Carsten Muller (BIOSI) and the group of Prof Ruedi Allemann (CHEMY) we have recently shown that it is a myrcene synthase linked to scent production in scented lines of Alstroemeria (Aros et al. 2012) (Fig 4)
Petals and leaves are considered to be of common evolutionary origins, and both senesce as their final stage in development. We therefore asked to what extent gene expression changes during senescence were shared between these two organs. We chose to use wallflowers (Erysimum linifolium) as our model, a member of the Brassicaceae in which flowers follow a clear developmental pattern lasting 8 days from bud to petal abscission (Fig. 5). Using microarrays we have shown that over half of the transcripts in an EST collection changed in expression in the same way in the two organs whereas expression of a class of chitinase related genes and GSTs was specific to petal senescence (Price et al., 2008) (Fig. 5). The role of reactive oxygen species (ROS) in petal senescence is far from clear (Rogers, 2012) and we are currently studying genes related to ROS and stress during petal senescence in both wallflowers ethylene-insensitive species.
Fig 5 - Stages of wallflower petal and leaf senescence, and functional analysis of genes specifically up-regulated during petal senescence (Price et al. 2008)
C) Post-harvest senescence
As part of an FP7-EU project including 14 partners from 7 member countries we are investigating new tools to improve safety and quality in ready to eat (RTE) salads and fruit salads (http://www.quafety.eu/). RTE salads are taking an increasing market share of fresh fruit and vegetable sales especially in Northern Europe. However they have a very short shelf life and, if handled inappropriately, can become contaminated with pathogenic organisms posing a serious health risk to consumers. Our project aims to improve quality and safety by providing new tools to evaluate quality and improve safety. In Cardiff I collaborate with Dr Carsten Muller on this project and we are using state of the art thermal desorption gas chromatography mass spectrometry (TD-GC-MS ToF) to detect volatile organic compounds that can be applied as markers for quality and safety of fresh cut produce. We are focusing on a leaf model: rocket salad (Diplotaxis tenuifolia and Eruca sativa) and a fruit model: cantaloupe melons. Our TD-GC-MS ToF work is in collaboration with Markes International. We have shown that VOC profiles of melon pieces are affected by the degree of processing (Fig 6)
Fig 6 - Use of volatile profiles as markers for the effects of processing. Small (S) and large (L) cut sizes were clearly discriminated on the basis of VOCs whereas the medium cut size (M) overlapped both.
2) Effects of stress on cell division
Plants are subject to numerous stresses including DNA damaging agents such as uv, soil pollutants and saline environments. Many of these agents cause an arrest in cell division until favourable conditions allow growth to resume. In collaboration with Dr Dennis Francis, Prof M Beatrice Bitonti and Dr RJ Herbert (University of Worcester) we have been studying genes that regulate these responses. DNA damage induces a cell cycle checkpoint arresting cells at G2/M One of the key regulators of this process in plants is WEE1 kinase which inactivates the the cyclin dependent kinase (CDK) by phosphorylation (Sorrell et al., 2002). We have recently shown using a yeast-two hybrid screen that Arabidopsis WEE1 interacts with proteins involved with proteasome-mediated degradation. Furthermore, the Arabidopsis WEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls (Cook et al., 2013) (Fig 7).
Fig 7 - Arabidopsis roots expressing AtWEE1-GFP treated with the proteasome inhibitor MG132 showing that inhibition of proteasome action inhibits the degradation on WEE1 (Cook et al, 2013)
In other eukaryotes CDC25 is a phosphatase that releases the block on cell division imposed by WEE1. A full length CDC25 is not present in higher plant genomes. However when the fission yeast gene Spcdc25 is expressed in plant cells it affects cell division, root development. These effects may be mediated by an increase in ethylene levels and changes in the cytokinin/auxin ratio (Spadafora et al., 2012)
We are also interested in how abiotic stresses such as salinity affect cell division and callus growth. Salinity is a major abiotic stress that limits plant productivity. Plants respond to salinity by switching on a coordinated set of physiological and molecular responses that can result in acclimation. We focussed on Medicago truncatula an important model legume species to test whether acclimation could enhance NaCl tolerance in calli. By the end of the 23 month experiment, calli were tolerant to 150 mM NaCl and showed enhanced expression of genes linked to cell division (such as WEE1)and salt stress Fig 8 (El Maghrabi et al., 2013).
Fig 8 - Expression of WEE1 in calli during the NaCI acclimation experiment (months in brackets) and representative calli by the end of experiment (El Maghrabi et al, 2013)
3) Mycelial interactions between competing fungi and fungal ecology
In collaboration with Prof Lynne Boddy, we have been using molecular approaches including microarrays to study gene expression during inter-species fungal interactions. Fungal interactions can result in deadlock or overgrowth by one of the competitors. Our microarray analysis indicates that changes in gene expression are related to the outcome (Eyre et al 2010; Fig. 9).
Fig 9 - Microarray analysis of gene expression changes in Trametes versicolor mycelium close to the interaction zone with competitors: Stereum gausapatum (Sg) Bjerkandera adusta (Bk) and Hypholoma fasciculare (Hf)
We have also focussed on ROS –related enzymes and shown that the activity of these enzymes is up-regulated close to the interaction zone between competing fungi in all interactions irrespective of outcome (Hiscox et al. 2010) (Fig. 10).
We are currently using high throughput sequencing approaches to understand the succession of fungal colonisation of fallen wood, and have recently been awarded a NERC grant ( in collaboration with Dr Dan Eastwood, Swansea) to further assess intermycelial interactions using RNAseq.
Fig 10 - laccase, manganese peroxidase and other peroxidases are up-regulated at the interaction zone between Trametes versicolor (Tv) and Stereum gausapatum (Sg) (visualised by staining) as well as in interactions between Tv and Bjerkandera adusta (B) and Hypholoma fasciculare (Hf) measured by enzyme activity
Other collaborative projects
A) Fungal taxonomy
In collaboration with Prof Lynne Boddy and Dr Martyn Ainsworth (Kew Gardens) we have been using targeted PCR primers in support of conservation efforts of rare UK fungi. We were able to show that Hericium species that fruit rarely are present as endophytes in the sapwood of many tree species (Parfitt et al, 2007). We have also used ITS sequencing to help to clarify taxonomic relationships within Hydnellum and Phellodon, which are often difficult to distinguish based on morphology(Ainsworth et al., 2009)(Fig 11), revealing the presence of cryptic species. Current work is aimed at extending our taxonomic understanding of these species.
Fig 11 - Phellodon melaleucus, B: Phellodon confluens, showing very similar morphology
B) Bacterial endophytes
In collaboration with Dr Esh Mahenthiralingam and Dr Colin Berry we are exploring further the movement of bacterial endophytes within plants and its biotechnological applications (Vidal et al., 2013 in press).
Staff currently associated with research:
David Parfitt, Julie Hunt, Jennifer Hiscox, Wafa Muftah (post-doctoral researchers on fungal populations and ecology)
Natasha Spadafora (post-doctoral researcher on post-harvest biology)
Basma Al Harbi (postgraduate on post-harvest stress biology)
Safaa AlFarsi (external postgraduate student on alfalfa landraces)
Faiza Hamdani (visiting postgraduate student on salt tolerant species)
Adel Elmaghrabi (recent postgraduate on stress and cell division)
Faezah Mohd Salleh and Danilo Aros (recent postgraduates on floral development and senescence)
Gemma Cook (recent postgraduate on stress and cell division)
Other International Collaborations
Prof Piero Picciarelli (University of Pisa, Italy) on floral senescence
Dr Lien Gonzalez (University of Havan Cuba) on the cell cycle
Prof Diego Albani (University of Sassari, Sardinia, Italy) on the cell cycle
Prof M. Beatrice Bitonti (University of Calabria, Italy) on the cell cycle
Prof Antonio Ferrante (University of Milano, Italy) on stress responses
Dr Wan Liu (China) on stress responses