Abel, S.; Theologis, A. Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression Plant Journal 5, 421-427, (1994)
An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyl-transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.
Abel, S.; Theologis, A. A polymorphic bipartite motif signals nuclear targeting of early auxin- inducible proteins related to PS-IAA4 from pea (Pisum sativum) Plant Journal 8, 87-96, (1995)
The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS-IAA4/5 and PS-IAA6, that encode short-lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS-IAA4 and PS-IAA6 using progressive deletion analysis and site-directed mutagenesis is reported. A C-terminal SV40-type NLS is sufficient to direct the β-glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS-IAA4 and PS-IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR-X(2471)-(RSXRK)/(RK/RK). Both basic elements of this motif in PS-IAA4, (KR-X41-RSYRK), function interdependently as a bipartite NLS. However, in PS-IAA6 (KKNEKKR-X36-RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40-type monopartite NLS. The spacer-length polymorphism, X(2470), in respective bipartite NLS peptides of several PS-IAA4-like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS-IAA4-like proteins supports the proposed integrated consensus of NLS.
Wong, L.M.; Abel, S.; Shen, N.; de la Foata, M.; Mal, Y.; Theologis, A. Differential activation of the primary auxin response genes, PS-IAA4/5 and PS-IAA6, during early plant development. Plant Journal 9, 587-599, (1996)
The plant growth hormone auxin typified by indoleacetic acid (IAA) transcriptionally activates early genes in pea, PS-IAA4/5 and PS-IAA6, that are members of a multigene family encoding short-lived nuclear proteins. To gain first insight into the biological role of PS-IAA4/5 and PSIAA6, promoter-β-glucuronidase (GUS) gene fusions were constructed and their expression during early development of transgenic tobacco seedlings was examined. The comparative analysis reveals spatial and temporal expression patterns of both genes that correlate with cells, tissues, and developmental processes known to be affected by auxin. GUS activity in seedlings of both transgenic lines is located in the root meristem, sites of lateral root initiation and in hypocotyls undergoing rapid elongation. In addition, mutually exclusive cell-specific expression is evident. For instance, PS-IAA4/5GUS but not PS-IAA6GUS is expressed in root vascular tissue and in guard cells, whereas only PS-IAA6GUS activity is detectable in glandular trichomes and redistributes to the elongating side of the hypocotyl upon gravitropic stimulation. Expression of PS-IAA4/5 and PS-IAA6 in elongating, dividing, and differentiating cell types indicates multiple functions during development. The common and yet distinct activity patterns of both genes suggest a combinatorial code of spatio-temporal co-expression of the various PS-IAA4/ 5-like gene family members in plant development that may mediate cell-specific responses to auxin.
Ticconi, C.A.; Delatorre, C.A.; Lahner, B.; Salt, D.E.; Abel, S. Arabidopsis <span style="font-style: italic;">pdr2</span> reveals a phosphate-sensitive checkpoint in root development Plant Journal 37, 801 - 814, (2004)
Levy, M.; Wang, Q.; Kaspi, R.; Parrella, M.P.; Abel, S. Arabidopsis IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense Plant Journal 43, 79 - 96, (2005) DOI: 10.1111/j.1365-313X.2005.02435.x
Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. To uncover regulatory mechanisms of glucosinolate production, we screened Arabidopsis thaliana T-DNA activation-tagged lines and identified a high-glucosinolate mutant caused by overexpression of IQD1 (At3g09710). A series of gain- and loss-of-function IQD1 alleles in different accessions correlates with increased and decreased glucosinolate levels, respectively. IQD1 encodes a novel protein that contains putative nuclear localization signals and several motifs known to mediate calmodulin binding, which are arranged in a plant-specific segment of 67 amino acids, called the IQ67 domain. We demonstrate that an IQD1-GFP fusion protein is targeted to the cell nucleus and that recombinant IQD1 binds to calmodulin in a Ca2+-dependent fashion. Analysis of steady-state messenger RNA levels of glucosinolate pathway genes indicates that IQD1 affects expression of multiple genes with roles in glucosinolate metabolism. Histochemical analysis of tissue-specific IQD1::GUS expression reveals IQD1 promoter activity mainly in vascular tissues of all organs, consistent with the expression patterns of several glucosinolate-related genes. Interestingly, overexpression of IQD1 reduces insect herbivory, which we demonstrated in dual-choice assays with the generalist phloem-feeding green peach aphid (Myzus persicae), and in weight-gain assays with the cabbage looper (Trichoplusia ni), a generalist-chewing lepidopteran. As IQD1 is induced by mechanical stimuli, we propose IQD1 to be novel nuclear factor that integrates intracellular Ca2+ signals to fine-tune glucosinolate accumulation in response to biotic challenge.
Chutia, R.; Abel, S.; Ziegler, J. Iron and Phosphate Deficiency Regulators
Concertedly Control Coumarin Profiles in Arabidopsis thaliana Roots
During Iron, Phosphate, and Combined Deficiencies Front Plant Sci 10, 113, (2019) DOI: 10.3389/fpls.2019.00113
Plants face varying nutrient conditions, to which
they have to adapt to. Adaptive responses are nutrient-specific and
strategies to ensure supply and homeostasis for one nutrient might be
opposite to another one, as shown for phosphate (Pi) and iron (Fe)
deficiency responses, where many genes are regulated in an opposing
manner. This was also observed on the metabolite levels. Whereas root
and exudate levels of catechol-type coumarins, phenylpropanoid-derived
2-benzopyranones, which facilitate Fe acquisition, are elevated after Fe
deficiency, they are decreased after Pi deficiency. Exposing plants to
combined Pi and Fe deficiency showed that the generation of coumarin
profiles in Arabidopsis thaliana roots by Pi deficiency considerably
depends on the availability of Fe. Similarly, the effect of Fe
deficiency on coumarin profiles is different at low compared to high Pi
availability. These findings suggest a fine-tuning of coumarin profiles,
which depends on Fe and Pi availability. T-DNA insertion lines
exhibiting aberrant expression of genes involved in the regulation of Pi
starvation responses (PHO1, PHR1, bHLH32, PHL1, SPX1) and Fe starvation
responses (BRUTUS, PYE, bHLH104, FIT) were used to analyze the
regulation of the generation of coumarin profiles in Arabidopsis
thaliana roots by Pi, Fe, and combined Pi and Fe deficiency. The
analysis revealed a role of several Fe-deficiency response regulators in
the regulation of Fe and of Pi deficiency-induced coumarin profiles as
well as for Pi deficiency response regulators in the regulation of Pi
and of Fe deficiency-induced coumarin profiles. Additionally, the
regulation of Fe deficiency-induced coumarin profiles by Fe deficiency
response regulators is influenced by Pi availability. Conversely,
regulation of Pi deficiency-induced coumarin profiles by Pi deficiency
response regulators is modified by Fe availability.