Longer text submission Ted B
8. Biographical Information
My full CV is online at http://virtualplant.ru.ac.za/Research/CV/Bothacv.htm (or at
http://tinyurl.com/7w3nkvx) where a detailed biography as well as direct access to many
papers is available. Where relevant specific papers are referenced as [15] etc. Specific
highlighted
conferences
may
be
accessed
at
this
URL
:
http://virtualplant.ru.ac.za/Research/CV/Internat-Conf.htm
My research career started at the University of Natal in Pietermaritzburg in the early 1970’s
where I was fortunate to be exposed to an environment where enquiry and exploration was
encouraged by my supervisor Prof CH Bornman. He successfully in getting one of the first
TEM and SEM microscopes into Southern Africa and I was immediately excited by the
opportunities that these instruments presented. I was interested in a number of issues – aphid
feeding and phloem soon coming to the fore (see [7-13]). Once completed I started a teaching
and research career at the University of Fort Hare where I was fortunate to be able to motivate
for good microscope facilities, culminating in the establishment of a first class electron
microscope unit. The UFH TEM allowed me to start my detailed study of plasmodesmata, and
to explore these fascinating structures, especially on their distribution and structure (see [19],
[22-23]. I was particularly lucky in that a long-term relationship started with Prof Ray Evert,
and many visits including several happy sabbaticals took place & my interests were fostered
and encouraged at all times. My collaboration with RHM Cross started at that time and
regular trips to Rhodes to use the TEM- this resulted in a long collaboration, with a number of
research articles dealing with grass leaf anatomy and plasmodesmatal biology (see [13], [2627] [35] [38] [39-42]. This collaboration continued until Mr. Cross retired. The UFH
microscope had a sophisticated EDAX facility – primitive as this would seem now, it resulted
in the first real proof of vascular transport and localization of heavy metals in plants (see [1416]). The interest was the ultimate location of the probes. After my move to Rhodes, a long
association with other people working in plasmodesmata commenced, staring with the
conference at Cognac, France in 1990, up to Pitlochry in Scotland, in 2006. No further longdistance flying occurred has been a problem for some years due to medical treatment for DVT.
Through the past 5-6 years I have developed techniques which are now helping to answer
questions about the complexities of the solute exchange pathways in plant leaves. This has
been challenging, yet rewarding. Challenging as there are severe limitations to microscopy
(particularly confocal) as none of the instruments available in South Africa are really suitable
for the probes we use, especially in green plants given that chlorophyll spoils the party with
such an all-encompassing emission spectrum, which can and does interfere with results on
other brands of instrument. rewarding when results are obtained. The ideal system exists at
Canberra and may yet exist at Fort Hare, should we successfully motivate for a Leica SP5
confocal.
The field that I choose to study (broadly, plant structure function) is not particularly popular,
anywhere. An excellent example of this is that the most recent Plasmodesmal Biology
Congress in Sydney, attracted only 36 speakers, with only two presentations classified as
strictly pd structure! This is not unexpected as many of these speakers are also active in the
assimilate transport field as well, which whilst it is a bigger group, it has many fringes which
are not strictly related to plant cell structure-function. I will argue that a small biological
specialist interest field attracts few citations.
I have developed techniques to allow study of the uptake fluorophores via the cut ends of
leaves specifically in the transpiration stream. The first results were presented at the Harvard
Forest meeting in 2002, also a related book chapter and then refined (presented at Bayreuth
meeting in 2003 (presented by A van Bel, as I could not travel due to teaching commitments).
I have received the SA Association of Botanists Silver Medal, been recogniosed as a
distinguished teacher, served on many NRF grant and assessment panels, and am on the
editorial board of the SA J Botany, where the editors ensure high quality outputs. I also
referee for Journal of Experimental Botany, Annals of Botany, Plant Biology and Plant
Growth Regulation, on a regular basis. I have also served on the Board of trustees of the
Albany museum. Details of this and other awards are available in my online CV.
Given the fixations with citation indices, I believe it is important to ‘level the playing fields’.
Working in a large field such as plant virology should yield proportionally much higher
citations than does the field I work in. Given the small size of the field, one does not expect
huge citation numbers or large h-indices. For example, searching all available databases for
‘plasmodes*’ yields 12 papers for Botha; the average h-Index of these is disappointingly, 9.
However, the total field has only 44 citations; the total h-Index in this field is 32. Using the
keyword ‘plasmodes* AND ultrastruct* yields only 29 papers, with an h-Index that is 18
world-wide; I have two of these papers, of which one has 36 and the other, 37 citations. I
believe that this is credible evidence that my work has an acceptable level of exposure
Significantly, the person who is credited with discovering major components of
plasmodesmata, A.W. Robards, has received only 56 citations in all, for his Nature paper,
published in the early 1960’s and an amazing 130 with W.J. Lucas for their 1990 review,
which was far broader than structure alone but covered plasmodesmata and virus and
molecular trafficking – together a really hot topic!
11. Best Student Outputs
MS. ELIZABETH ADE-ADEMILUA (Ph.D. (awarded 2006). She assessed PI as a growth
indicator in bean plants under controlled environment., the pity, as stated by her externals,
Four published papers from the thesis, 6 citations, confirming the view of externals that this
“mathematical predictor of plant growth is (will be) largely ignored by those who should use
it, plant scientists!”
MR. S.A. SAHEED 2005: (Ph.D awarded, 2008). Saheed’s thesis was a first quantification of
damage induced by the Bird-Cherry oat aphid (BCA)and two RWA biotypes, he calculated
10-15% crop loss. Controlled environment was used to model colony effects and growth rates
and a study of short- and long-term feeding on the vascular system. His research highlights
severe damage caused to xylem by these aphids as well, which results in wilting & chlorosis.
(see 2: VASCULAR TRANSPORT under the overview). He undertook a postdocl visit and
presented at an international meeting (see http://virtualplant.ru.ac.za/Research/CV/InternatConf.htm, for details). His four papers have already attracted 9 citations.
MR. J. MAHBOOB 2008 (PH. D. awarded 2011). This thesis examined the long-term effects
of damage inflicted by RWA and BCA on . The study used several known US resistant
barley lines and to compare these to non-resistant and resistant South African varies.
Fluorescence and TEM used to assess damage. He also modelled aphid breeding rates under
normal and elevated CO2. He demonstrated that two RWA biotypes (SA2 and SA3) were
more virulent and damaging to host plants. Elevated CO2 increased growth (aphid popn &
plant size, and nitrogen loss resulted in early plant death. He has a total of 9 publications, two
from his thesis (2011) have been published and others are in preparation.
MR. IBRAHEEM OMODELE Co-Supervisor with Prof G. Bradley. (Ph.D. awarded 2010
University of Fort Hare). Omodile worked at Fort Hare under Prof Bradley. The cosupervision of this thesis involved ensuring that he was able to characterize the distribution
and upregulation of the rice sucrose transporter OsSUT-1. His work confirms an hypothesis
that the observed distribution of OsSUT in xylem parenchyma was related to retrieval of ‘lost’
solutes (due to cell damage or aphid feeding). We applied a new fluorescence approach using
Imagene-Green instead of GUS expression to be able to localize the upregulated transporter.
He has two papers, one of which I co-author one and a major paper ready for submission
dealing with the upregulation of OSSUT-1 due to aphid infestation.
13: Brief description:
Please
Note!
References
(eg
[15])
http://virtualplant.ru.ac.za/Research/CV/Peer-Reviewed.htm
are
available
at
I have continued to focus my research in strict terms, on an in-depth study of transport
systems in vascular plants. This continues to be driven by curiosity and a desire more fully
understand/comprehend plant cell transport interactions and the processes that take place in as
a result, but primarily in leaves. What controls transport? What inhibits transport of certain
substance or particles or molecules? The two functional transport compartments in vascular
systems are for all intents and purposes, separate – or are they? There is a need to assess the
relative importance and interaction between two dynamically, functionally and structurally
different systems. To this end, we make use of TEM, fluorescence brightfield and when
possible, confocal microscopy, to examine cell functional relationships within vascular
systems of leaves. The information and new knowledge that is gained from past work, fuels
future and parallel research. Information gained in the several in-depth studies about the
unique structure of plasmodesmata in especially the grasses, drives my continued passion for
understanding intercellular communication and the implication of functional plasmodesmata
in this process. A thorough and rigorous study remains core to more clarity about cell
(inter)relationships at the vascular level. Additionally, the control systems that maintain
phloem and xylem function within the constraints of the vascular bundle are also critical
components in this overarching interest in cell communication. Close and careful examination
of feeding and probing and the subsequent damage sustained by the vascular system, resulting
from the ravages caused by aphid probing and feeding on vascular tissues. Here, we have a
unique opportunity to examine the effect of salivation , probing and feeding on the two
principal transport systems – phloem as well as xylem are core studies on their own. Aphids
thus are important tools and studies based on cell examining cell damage, are highly relevant
to my overall interest in plant vascular systems. Cell puncturing and salivation by aphids,
offers a unique opportunity to examine in situ cell damage and disruption to transport systems,
as vascular disruption caused by probing and especially stylets and saliva, offers unique
opportunities to investigate regulation, disruption, functionality and dysfunction at the cellular
level, of both transport systems independently and as an integrative whole. They therefore
form one of the three legs on which the foundation of my research depends. The three core
areas of my research and which are most relevant to this review cycle are (1) plasmodesmata,
[form, function, structure]; (2) vascular transport systems [role of xylem in solute exchange,
role of xylem parenchyma in endo- and exocytosis, uptake via apoplast and symplast and fate
(phloem loading)] (3) vascular system damage/disruption due to aphid feeding [stylet related
physical damage (leakage), saliva & disruption of pit membranes, loss of solute flow].
1 PLASMODESMATA AND THEIR ROLE IN INTERCELLULAR TRANSPORT: Many
of the scientists interested in plasmodesmata are more involved in studies of virus movement
or molecular based studies. Few of us are involved in TEM investigations of structurefunction relationships in higher plant plasmodesmata (pd) or in documenting the variation in
structure and configuration visible. In one of the earliest authors Lopez-Sayez et al (1965)
wrote that “the continuous passage of the filaments of the endoplasmic reticulum from a cell
to its neighbours is still debatable”. AW Robards in his letter to Nature in 1963, first
described the tubule in the centre of plasmodesmata, and drew attention to its similarity to
cytoplasmic microtubules, naming it a desmotubule. This 1963 Nature paper is thus the first
record of the naming of the components of a plant plasmodesma. Yet surprisingly few
citations exist for this (56) yet the review by Lucas and Robards in 1990 has received at least
292 citations. This review was published subsequent to the NATO conference in1989, which
focussed attention and drew on the similarities between gap junctions in animal and human
cells, and plant plasmodesma. A number of papers and posters were presented at the Cognac
Conference in 1990. As far as I was concerned, plasmodesmatal biology had arrived! Much
has been researched and written about pd, and several wonderful international conferences
have been held over the years, where their structure and functional importance, as well as
their importance as gateways for cell to cell trafficking has been debated and often quite
heatedly! So what is so special about these cell connections? I believe that it is their wide
range of structural variation that makes them so utterly fascinating. The variations that are
seen in structure are likely related to the particular function that these plasmodesmata (pd)
have, at the interfaces they cross. For example the plasmodesmata in the Kranz-mesophyll
bundle sheath interface (KMS-BS) in C4 grasses are constricted where these cross the suberin
lamella, as too, are the pd at the vascular parenchyma interface to intermediary cells; but for
different purposes. In the KMS-BS interface, the constrictions reflect the narrow, primary
origin of these numerous pd, constricted during the laying down of the suberin lamella; in the
intermediary cells, the inner constriction acts as a ‘valve’ preventing higher molecular-weight
sugars from leaking back through to the BS, those in the KMS-BS are still large enough to
allow the passage of sugars. How do we know this? Through a combination of a structured
and detailed electron microscopy investigation which included statistically robust frequency
determinations, coupled in many instance with other experiments using transportable
fluorescent probes and or other molecular studies to confirm the structural studies. Answers to
these questions are really only possible through rigorous experimental approaches, using
TEM as the primary means of investigation. The changes in structure (related to possible
functional changes) which have been explored previously and which I continue to explore
makes pd as interesting now, to me, as they must have been to early researchers such as the
information published by Robards, (1963) and Lopez-Saez et al. 1966).
2 VASCULAR TRANSPORT: Early data from experiments involving the study of the uptake
of heavy metals such as lead and lanthanum, as well as the complex, Prussian blue, suggested
that there could be an exchange pathway. Unfortunately much of this information was ignored
at the time, because of the focus on the potential pathway through the intercellular spaces and
middle lamella region. Later, the introduction of fluorescence microscopy coupled with the
use of symplasmically directed probes, demonstrated that there was an interchange pathway
between xylem vessels and surrounding parenchyma tissue. Furthermore, we suspected that
this would or could be, important in transfer and most probably, also possibly in the
regulation of solute movement. We knew from previous studies (see [19], [21], [33] that this
interface involved several possible pathways, apoplasmic (diffusion driven) as well as
symplasmic, (membrane-controlled) transport between xylem vessels and their adjoining
parenchyma cells. Early experiments were frustrating, not from the point of view of probe
delivery, but technical issues needed to be overcome relating to sectioning and prevention of
diffusion of the probes, from cut sections. This data was presented at the Harvard Forest
meeting and I was invited to contribute a chapter in the book published by Holbrook and
Zwieniecki in 2005. A sabbatical offer at Plant Industry in Canberra, resulted in the
publication of the first in what will be a series research papers (see Botha et al 2008, [55]
(http://tinyurl.com/6o6gsnt ). involved an in depth confocal and fluorescence microscope
study of the movement of a number of xenobiotic probes fed via the transpiration stream in
mature rice leaf blades. This study revealed that apart from providing passageway for water
efflux from the xylem vessels to their parenchyma cells, that these cells channelled cleaved 5,
6-Carboyfluorescein diacetate, to the adjacent xylem parenchyma, via the pit membrane,
which is lined by the plasmamembrane on the inner parenchyma cell side. Given that these
vascular parenchyma cells contained large mitochondrial populations, numerous dictyosomes,
well-developed endomembrane complexes and that there were numerous vesicles in close
proximity to the pit membrane, we surmised that endocytosis could be occurring at this
interface. To test this, we applied Lucifer Yellow and Texas red-labelled 10kDa dextran to the
transpiration stream, which ended up in vascular parenchyma cells, confirming endocytosis as
there was no way that these membrane-impermeant probes could have offloaded from the
xylem via the pit membranes, other than through endocytotic transmembrane transfer. A
further important point, which is picked up on later in the phloem transport section, is that
accumulation within the thick-walled sieve tubes, but not in thin-walled sieve tubes,
confirmed a symplasmic phloem-loading pathway, between xylem parenchyma and thickwalled sieve tubes, but not between xylem parenchyma and the thin-walled sieve tubes. The
discovery or confirmation of an endocytotic pathway is under investigation (discussed under
future research).
3. APHID FEEDING STUDIES: As mentions, one of the drivers for continuing with basic
aphid feeding experiments is to be able to evaluate the damage cased and sustained as a result
of the aphids’ activities. Collectively, our studies showed that aphid feeding caused severe
damage no only to the phloem, but also to xylem elements. This has been reported in several
papers, that have demonstrated this destructiveness (see de Wet et al., 2007,
http://tinyurl.com/7n8the2 ), Saheed et al 2007, http://tinyurl.com/7c8qn2m, and Saheed et al
2007 (http://tinyurl.com/7nmcy9q ), and later by Jimoh et.al
(2011,
http://tinyurl.com/6ocoqsc ).
Information gained from the aphid-related study, has informed the other interest areas also,
specifically with respect to the interrelationship between the components of the vascular
system. The aphid work is on-going, and demands TEM as well as fluorescence microscopy. I
continue to make use of detailed TEM as well as wide-field & confocal microscopy to study
the xylem-parenchyma interface & of transport across this interface. I have explored a number
of species and encouragingly, all have yielded similar results. The xylem-xylem parenchyma
interface is an active region in which endocytosis has been demonstrated at the light,
fluorescence confocal and TEM level. Much of this information is near publication stage,
with most of it in collaboration with Prof Bradley, Dr Edwards and our shared students.
14: Self-Assessment
NOTE: Numbers in square brackets [ ] refer to papers in my CV. I think I could have
achieved more in the past 8 years than I have been able to. Achievement is based and
dependent on a number of things, most important of which are equipment availability (and
reliability) and free time to travel to complete work. I stated in my previous evaluation
assessment that microscopy – TEM, SEM fluorescence and confocal are essential. Several of
the research questions that I have asked in previous grant applications are absolutely reliant
on time and effort. Again, this is why ‘explorative microscopy’ is not popular and will never
draw in large research groups at is not quite as fashionable (arguably, informative?) as others.
The focus over the past 8 years has been dictated to a large extent by these constraints as well
as enticing students and maintaining interest in doing this kind of research, into the laboratory.
I was indeed very fortunate in attracting Dr Lin Liu, to Rhodes to take up the first of two Post
Doctoral positions. Lin’s infinite patience with students (training them when free, to the art
and finer points of sectioning and TEM observation) as well as out very busy joint schedule,
which meant a great deal of time was dedicated to sectioning and viewing and then discussing
results. His insight into what was needed was a fantastic help and we made excellent progress.
Our focus was plasmodesmata initially and specifically, those in Cyperaceae. This
information was delivered first as a paper, at the Asilomar Plasmodesmatal biology
conference and subsequently as a book chapter in the 2005 book, ‘Plasmodesmata’ edited by
KJ Oparka. This work provided additional useful information concerning the substructure of
the intriguing plasmodesmata in general but more importantly that the Cyperaceae had rather
unique structures.
Saheed and Jimoh were interested in the aphid feeding projects. These studies, without doubt,
contributed significantly to us gaining a better understanding of aphid-inflicted cell damage.
This also contributed to the ‘I wonder what will happen if…’ approach that I like to take
frequently in my research approach. Resulting from this kind of approach, we were able to
answer the question as the what happened to the xylem when aphids drank water? It turned
out to be a significant finding. When these aphids drank water, stylet insertion into the xylem
vessels was not without severe physiological perturbation. The chance of transient embolism
as a physiological perturbation could not be discounted – the result being that air bubbles in
the xylem would impede flow, the end result being wilting (a commonly noticed aphid
feeding-related phenomenon). This wilting occurs because the xylem is, under normal
circumstances, a ‘leaky’ system thus ensuring a supply of water and nutrient to leaves.
Embolisms were not evident, but saliva was! How did this happen? Once punctured the
aphids clear the stylet food canal of debris, before drinking. In the process, watery saliva is
also ejected. We showed evidence of this saliva at the TEM level, also of the extent of the
damage it caused when deposited particularly in the hydrolysed pit membranes of halfbordered pit pairs. This effectively, pu the bathplug in, as the saliva plugged pores, resulting
in leaf wilt and chlorosis [54, 62]. These papers as well as conference proceedings,
contributed to my thinking on the role of the xylem in solute exchange. Substantive effort was
made to develop techniques to enable us to examine phloem-mobile xenobiotics, through
flap-feeding techniques which were applied by students [44,48, 57,& indirectly in 62]. An
invitation to participate at the Harvard conference led to the book chapter in Holbrook and
Zwieniecki (2005) and a decision to travel to Australia to collaborate at the CSIRO. The
Australian collaboration was highly successful. Apart from the excellent data obtained using
the Leica confocal, we fixed tissue for later TEM investigation, as I was interested in seeing
where efflux of xenobiotics was taking place, and to examine the ultrastructure of the xylemassociated parenchyma cells. This collaboration gave rise to what I think is a key paper [55]
which clearly demonstrated that endocytosis was the only possible explanation for transfer
from the xylem. The second paper is in near final form now. I believe that the results obtained
though not numerous, will have impact in plant biology and that these results will inform
aspects of plant structure and function, which will possibly have fundamental and applied
outcomes. Time will tell.
15 On-going and planned
Please follow the link at http://tinyurl.com/7nfzmgf for more information about on-going
research
A big plus is that I have no further undergraduate teaching responsibilities, and thus can
devote a great deal of time to research. I have a number of major research efforts underway
mostly near-completion. In a previous application I stated that my underlying motivation for
doing research was simply, curiosity. Curiosity in this context, extends to carrying on with the
cell-cell transport & xylem to phloem interchange programme. Much of my research will be
strongly centred here at Rhodes, and the focus of this ‘home base’ research will be unaltered.
I am scheduled to travel to Australia again, this year to continue working on this problem with
Dr White as a principal co-investigator from Plant Industry and Dr Edwards from entomology.
The plan is to track some of the more difficult xenobiotic probes using the Leica confocal
systems, and by adding aphids to the mix, to study the effect of salivary excretion into the
xylem on transport thereby to elucidate the potential pathway(s) available. I think this will be
a ground-breaking study, bringing together experienced microscopists, entomologists and
molecular biologists, all collaborating on one focussed programme.
AIM: In broad terms, the overarching aim is to explore and understand control and regulation
of transport across the xylem to phloem interface. This will primarily be done on rice and for
the aphid work, whatever aphid is available (legally) in Canberra, on lupin or other plants of
interest. A range of techniques, including molecular markers, quantum dot and associated
microtechniques, including aphid feeding, (nice lab method to study cell damage and
perturbations that they do inflict) will be used. Aphids will provide causal feeding-related
effects to study effects on endocytosis, as well as calcium signalling. In order to be able to
answer the principal aim requires that I address a number of questions.
Several questions still need more complete investigation. What pathways are involved in
retrieval across the xylem exchange interface and what effect (transient/permanent) do aphids
impose? 2. What is the ultimate fate of the xenobiotics? Are these cytoplasmicallyconstrained or are they accumulated in the vacuole? 3. re there circumstances which favour
this exchange process? 4. Can we elucidate the structural and physiological barriers in place
which may regulate transfer across the xylem exchange interface? 4. To explore the
catastrophic effect of aphid feeding on the phloem can solutes find alternative routes?
In addition to this I will be taking up a part-time position at Fort Hare as Adjunct Professor in
Botany and Plant Biochemistry and will be responsible for a number of cogent research
students and the development of molecular microscopy techniques at Fort Hare. Naturally, the
aim is to improve and strengthen the biological sciences at Fort Hare, which will beneficiate
any future collaborative research with anyone interested in collaborating regionally and
nationally. Finally, I plan to refocus the aphid work to fit within the climate change
framework programme, to be centred at Fort Hare. I will direct research (structure and
functional based) towards a better understanding of the effects of short-term and long term
exposure to elevated (450 and 550 ppm) CO2 – Apart from the pilot studies and work being
done in my lab here at Rhodes, to model elevated CO2 effects, little is actually known about
the effects on plant transport system when aphids are added to the mix. This programme will
be funded for the most part through my collaboration at Fort Hare.