Plant Molecular Biology

Stress Response to Different Concentrations of NaCl Analysis of Root Length and Protein Expression on Wild Type Arabidopsis thaliana

by Ravi Dinakar

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Authors:

Samantha Giffen1*and Julie Nowicki2
Student1, Teacher2: Biotechnology High School, 5000 Kozloski Road, Freehold, NJ 07728
*Correspondence: samantha_giffen@bths.mcvsd.org

Published in The Journal of Experimental Secondary Science (www.jes2s.com)

The purpose of this experiment was to examine the stress
response of wild type Arabidopsis thaliana to... more The purpose of this experiment was to examine the stress
response of wild type Arabidopsis thaliana to treatment
with different sodium chloride concentrations. Previous
research has shown that sodium chloride induces a
stress response from plants in both a physical sense and
molecular sense. The hypothesis was that the plants would
exhibit a number of different stress responses including
shorter root length and an increased production in specific
stress- responsive proteins. The hypothesis was tested
by placing germinated Arabidopsis seedlings onto MS
(Murashige and Skoog) agar plates with varying sodium
chloride concentrations. While the seedlings grew on the
plates with sodium chloride, root length was measured.
After root length measurements were completed, specific
plants from each concentration were subjected to protein
analysis through SDS-PAGE to compare the banding
patterns of protein expression for each treatment group
versus the control. The importance of this study was to
better understand how plants tolerate different levels of
sodium chloride. Root length analysis showed that higher
concentrations of sodium chloride dramatically inhibited a
plant’s growth. SDS-PAGE analysis showed a protein band
that was present in the control plants and was not present
in the plants that grew in an added concentration of sodium
chloride. These results show that the plants have specific
stress responses to sodium chloride including shorter root
length and a decrease in the production of an unkown
protein.

The Effect of Soil Nitrogen Levels on Thigmotropic Responses in the Venus flytrap, Dionaea muscipula

by Ravi Dinakar

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Authors:

Lauren Howard1,*, James Philps1,*, Jamie Shenk1,*, Min Jae Yoo1,*, and Geoffrey R. Tanner2**
Student1, Teacher2: Department of Biology, Phillips Academy, Andover MA, 01810
*These authors contributed equally to this work.
**Correspondence: gtanner@andover.edu

Venus flytraps are perhaps the world’s most well-known
carnivorous plant. They rely on small insects to... more Venus flytraps are perhaps the world’s most well-known
carnivorous plant. They rely on small insects to acquire
nitrogen, as the swampy environment that they inhabit
in southeastern United States’ Carolina coastal plains
is insufficient in nitrogen. We studied the effect that soil
nitrogen levels have on the flytrap’s carnivorous behavior.
We manipulated soil nitrogen levels to produce nitrogensufficient
and nitrogen-deficient growth conditions for
Venus flytraps. We then compared the closure time of
the snap traps of the plants in nitrogen-sufficient and
nitrogen-deficient soil, following touch stimulus. We
found that flytraps in nitrogen-rich soil exhibited longer
closure times. We concluded that, because the plants were
grown in a nitrogen-rich environment, they did not require
supplemental insect nitrogen, and were possibly conserving
energy by responding less vigorously to touch stimulus.

Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction

by Mario Costa

Sílvia Coimbra, João Almeida, Vítor Junqueira, Mário Costa and Luís Gustavo Pereira

Abstract
Some of the most important changes that occur in
plants during sexual reproduction involve the... more Abstract
Some of the most important changes that occur in
plants during sexual reproduction involve the transition
from a sporophytic to a gametophytic type of develop-
ment. In this paper, these changes were evaluated for
Arabidopsis thaliana. The results obtained clearly show
differences in the pattern of distribution of specific
arabinogalactan protein (AGP) sugar epitopes, during
anther and ovule development. AGPs are hydroxyproline-
rich glycoproteins that are massively glycosylated and
ubiquitous in plants. The molecular mechanism of
action of AGPs is still unknown, mainly due to the
difficulties posed by the complex saccharide chains.
However, the complex structure of the sugar fraction
of AGPs makes them a potential source of signalling
molecules. The selective labelling obtained with AGP
mAbs JIM8, JIM13, MAC207, and LM2, during Arabi-
dopsis pollen and pistil development, suggests that
some AGPs can work as markers for gametophytic cell
differentiation. Specific labelling of the first gameto-
phytic cells in the pistil, the strong labelling of the
secretory cells of the embryo sac, the synergid cells,
and the labelling of the integument micropylar cells,
apparently outlining the pollen tube pathway into its
final target, the embryo sac, have all been shown. In
the anthers, the specific labelling of gametophytic
cells, and of the male gametes that travel along the
pollen tube, may indicate AGP epitopes acting as
signals for the pollen tube to reach its final destiny.
The specific labelling of cells destined to go into
programmed cell death is also discussed.

Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants.

by Mario Costa

Sílvia Coimbra, Mário Costa, Brian Jones, Marta Adelina Mendes, and Luís Gustavo Pereira.

Abstract
Arabinogalactan proteins (AGPs) are structurally complex plasma membrane and cell wall proteoglycans... more Abstract
Arabinogalactan proteins (AGPs) are structurally complex plasma membrane and cell wall proteoglycans that are
implicated in diverse developmental processes, including plant sexual reproduction. Male gametogenesis (pollen
grain development) is fundamental to plant sexual reproduction. The role of two abundant, pollen-specific AGPs,
AGP6, and AGP11, have been investigated here. The pollen specificity of these proteoglycans suggested that they
are integral to pollen biogenesis and their strong sequence homology indicated a potential for overlapping function.
Indeed, single gene transposon insertion knockouts for both AGPs showed no discernible phenotype. However, in
plants homozygous for one of the insertions and heterozygous for the other, in homozygous double mutants, and in
RNAi and amiRNA transgenic plants that were down-regulated for both genes, many pollen grains failed to develop
normally, leading to their collapse. The microscopic observations of these aborted pollen grains showed
a condensed cytoplasm, membrane blebbing and the presence of small lytic vacuoles. Later in development, the
generative cells that arise from mitotic divisions were not seen to go into the second mitosis. Anther wall
development, the establishment of the endothecium thickenings, the opening of the stomium, and the deposition of
the pollen coat were all normal in the knockout and knockdown lines. Our data provide strong evidence that these
two proteoglycans have overlapping and important functions in gametophytic pollen grain development.

Association study between the Gibberellic Acid Insensitive gene and leaf length in a Lolium perenne L. synthetic variety

by Abraham Escobar-Gutierrez

The Apoplastic Oxidative Burst Peroxidase in Arabidopsis Is a Major Component of Pattern-Triggered Immunity

by Arsalan Daudi

Plant Cell, 2012

Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel F, and Bolwell GP (2012) The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell (published online January 24th 2012)

Molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase

by Carlos Rodriguez-Caso

Development, characterization and use of microsatellite markers for germplasm analysis in date palm (Phoenix dactylifera L.)

by Hesam Arabnezhad

Highlights

► A novel set of microsatellite loci suitable for genotyping were developed from two enriched genomic libraries for Phoenix dactylifera L. ► The cluster analysis placed African date palms in a group different from Iranian and Iraqi genotypes. ► It seems that the domestication of African date palms have followed a different route than those grown in the Middle-East. ► The results suggested a significant relationship between genetic differentiation and geographical distances of date palm cultivars.

The present study was undertaken to assess genetic relationships among date palm (Phoenix dactylifera L.) genotypes... more The present study was undertaken to assess genetic relationships among date palm (Phoenix dactylifera L.) genotypes grown in different geographical regions by using newly developed simple sequence repeat (SSR) markers. Two SSR-enriched genomic libraries including repeat motifs (AG)n and (AAG)n were constructed in date palm. Based on DNA sequences of positive clones, 25 primer pairs were designed of which 22 pairs were able to detect polymorphism in 16 date palm cultivars from Iran, Iraq and Africa. The selected SSR primers amplified a total of 106 alleles with an average of 4.82 alleles per locus among the cultivars and the average values of He and PIC were 0.719 and 0.668, respectively. Neighbor-Joining cluster analysis based on Nei's genetic distance divided date palm accessions into three major clusters in agreement with their geographical origin. Cluster analysis significantly distinguished African cultivars from Iranian and Iraqi ones, suggesting that the domestication of African date palms have followed a different route than those grown in the Middle-East, an assumption which is supported by Mantel test and Bayesian analysis. The set of date palm SSR loci developed in this study could be an informative marker system for geographic partitioning and genotyping of date palm germplasm.

Anatomical and molecular identification of "guaco" Mikania glomerata e Mikania laevigata (Asteraceae), two important medicinal species from Brazil

by Carolina Lopes Bastos

Mikania glomerata Sprengel and Mikania laevigata Schultz Bip. ex Baker are native Brazilian lianas, popularly known as... more Mikania glomerata Sprengel and Mikania laevigata Schultz Bip. ex Baker are native Brazilian lianas, popularly known as guaco and mainly used as bronchodilators. Based on morphological and anatomical leaf features, preliminary data indicate substantial similarity between the two species, requiring further studies to increase the accuracy of their diagnosis. However, it is still unclear which of the two species is commercially sold. Therefore, in the anatomical analysis, three individuals of each species were treated according to the usual methodology for stem anatomy. Molecular analysis was performed on four specimens of M. glomerata and six of M. laevigata, using three plastid loci (matK, rbcL and trnH-psbA) and the ITS2 locus, which have been proposed as plant barcodes. Our results, including anatomical descriptions and statistically derived quantitative data, show that the anatomical structural standard is very similar between the species. Molecular data corroborate the morphological data in pointing to the total similarity between the two species observed in the loci used. Based on these results, we conclude that M. laevigata and M. glomerata could be unified in terms of nomenclature.

Establishing a time-scale for plant evolution

by John Clarke

Expression of Arabinogalactan Protein Genes In Pollen Tubes of Arabidopsis Thaliana

by Hugo Rafael Oliveira

Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites In Plants

by Arsalan Daudi

Plant Physiology, 2010

Nakagami H, Sugiyama N, Mochida K, Daudi A, Yoshida Y, Toyoda T, Tomita M, Ishihama Y, and Shirasu K (2010) Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants. Plant Physiology 153: 1161-1174.

Plant pathogens: how can molecular genetic information on plant pathogens

by Arsalan Daudi

Keynote Address, 2004

Hammond-Kosack K, Urban M, Baldwin T, Daudi A, Rudd J, Keon J, Lucas J, Maguire K, Kornyukhin D, Jing H, Bass C, and Antoniw J (2004) Plant pathogens: how can molecular genetic information on plant pathogens assist in breeding disease resistant crops. Proceedings of the 4th International Crop Sciences Congress, Brisbane, Australia.

Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

by Yuji Iwata

IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a... more IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

Rapid Cloning of Novel Genes and Promoters for Functional Analyses In Transgenic Cells

by Claudia Vickers

The availability of sequence information for thousands of genes for many organisms is currently unmatched by... more The availability of sequence information for thousands of genes for many organisms is currently unmatched by functional studies. A cost-effective and high-throughput cloning system for PCR products was therefore adopted to enable the rapid assessment of coding and promoter sequences in functional assays in transgenic cells. Unlike other systems that involve initial cloning into a specialized PCR fragment cloning vector, this method describes a rapid and cost-effective procedure for the amplification of a DNA fragment by PCR, its phosphorylation and its direct insertion into the vector of choice. Restriction enzymes are only required once for the preparation of the recipient vector, which is blunt-ended and dephosphorylated. No special primer designs (e.g. restriction enzyme sites or flanking homologous sequences) or subcloning steps are required. The turn-around time from source organism genomic DNA to new recombinant DNA is 24 hrs. It is particularly suitable for functional genomics projects or the generation of libraries from PCR products where a large number of fragments need to be cloned into the same vector. We have used this method to rapidly clone 72 full-length genes (ranging from 0.8 to 6.4 kb) and putative promoters (2 kb each) from Arabidopsis thaliana into plant cell expression cassettes for subsequent direct functional analyses in transgenic cells.

Genetic Structure and Regulation of Isoprene Synthase In Poplar (Populus Spp.)

by Claudia Vickers

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-d-erythritol... more Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-d-erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

A Novel Cis-Acting Element, ESP, Contributes to High-Level Endosperm-Specific Expression In An Oat Globulin Promoter

by Claudia Vickers

To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters... more To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5′ deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCATCATGT, was required for ES specificity and substantially contributed to expression strength of the␣AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.

Promoter Analysis of the Barley Pht1; 1 Phosphate Transporter Gene Identifies Regions Controlling Root Expression and Responsiveness to Phosphate Deprivation

by Claudia Vickers

Previous studies have shown that the promoter from the barley (Hordeum vulgare) phosphate transporter gene, HvPht1;1,... more Previous studies have shown that the promoter from the barley (Hordeum vulgare) phosphate transporter gene, HvPht1;1, activates high levels of expression in rice (Oryza sativa) roots and that the expression level was induced by up to 4-fold in response to phosphorus (P) deprivation. To identify promoter regions controlling gene regulation specificities, successive promoter truncations were made and attached to reporter genes. Promoters of between 856 and 1,400 nucleotides activated gene expression in a number of cell types but with maximal expression in trichoblast (root hair) cells. For shorter promoters the trichoblast specificity was lost, but in other tissues the distribution pattern was unchanged. The low P induction response was unaffected by promoter length. Domain exchange experiments subsequently identified that the region between –856 and –547 nucleotides (relative to the translational start) is required for epidermal cell expression. A second region located between 0 and –195 nucleotides controls root-tip expression. The HvPht1;1 promoter contains one PHO-like motif and three motifs similar to the dicot P1BS element. Analysis of promoters from which the PHO-like element was eliminated (by truncation) showed no change in the gene induction response to P deficiency. In contrast, mutation of the P1BS elements eliminated any induction of gene expression in response to low P. An internal HvPht1;1 promoter fragment, incorporating a single P1BS element, had an increased response to P deprivation in comparison with the unmodified promoter (containing three elements). Together these findings further our understanding of the regulation of the HvPht1;1 gene and provide direct evidence for a functional role of the P1BS element in the expression of P-regulated genes

Isoprene synthesis in plants: lessons from a transgenic tobacco model

by Claudia Vickers

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially... more Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.

A synthetic xylanase as a novel reporter in plants

by Claudia Vickers

Transient gene expression assays are often used to screen promoters before stable transformation. Current transient... more Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene (sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUSPlus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems