Journal of Undergraduate Research
Volume 5, Issue 1 - October 2003

Differential Expression Studies in Hydrilla verticillata

Nathan Bazinet, Srinath K. Rao, Julia Reiskind, Gonzalo M. Estavillo and George Bowes

INTRODUCTION

Plants exhibit three major types of photosynthesis: C3, C4 and CAM. C3 plants initially fix CO2 through the enzyme ribulose bisphosphate carboxylase/oxygenase (Rubisco). However, fixation of CO2 and O2 by Rubisco occur at the same active site. Thus there is competition between the two substrates, and when O2 reacts it reduces photosynthetic CO2 fixation. C4 plants circumvent this dilemma by using an additional C4 acid cycle that acts as a CO2“pump”, which, in conjunction with a specialized leaf structure (Kranz anatomy), results in a high CO2 concentration in the cells where Rubisco is located. This system enhances CO2 fixation. Hydrilla verticillata is a submersed, freshwater angiosperm that possesses the unique ability to induce a C4 mechanism without the specialized Kranz leaf anatomy of terrestrial C4 plants (Bowes et al, 2002). At ambient [CO2] Hydrilla exhibits C3 characteristics, but as CO2 becomes limited and temperatures rise, enzymes associated with the C4 cycle are up-regulated. This study employs the technique of differential display to identify differentially expressed genes in C4 Hydrilla leaves. Differential expression is a powerful technique that allows a rapid screening of RNA populations from biological systems (Liang and Pardee, 1992).

MATERIALS AND METHODS

Plant material and growth conditions


Hydrilla verticillata (L.f.) Royle plants were collected from Lake Oklawaha (Putman County, FL). Plants were washed and cut to 6 cm long sprigs and maintained in 5-L aquaria with a 25ÖC, 12-h photoperiod and a PPFD of 250 mmol m-2 s-1. From this stock, three shoots per tube were placed in 3.5 ¥ 20-cm test tubes containing 80 mL distilled water and incubated for up to 15 d under a 30ÖC/14-h photoperiod with a PPFD of 250 mmol m-2 s-1 and a 22ÖC scotoperiod. The water was changed every other day, and the pH and dissolved inorganic carbon were allowed to fluctuate with the metabolism of the plants. Samples were collected after 15 d and frozen in liquid nitrogen.

Enzyme extraction and activity measurement

Both extraction and activity determinations followed the procedures described in Magnin et al (1997). Protein was determined following the method of Bradford (1976).

Differential display

Liquid nitrogen frozen C3 and C4 plant samples were ground to a fine powder, and RNA was extracted according to RNeasy Plant Kit protocol, (Qiagen, USA). A DNase treatment step was included to remove chromosomal DNA contaminants. Approximately 15 mg of total RNA from each of the C3 and C4 tissues were used to isolate mRNA in a total elution volume of 60 μL per sample with the help of oligotex mRNA kit (Qiagen Inc., CA, USA). First strand cDNA synthesis was performed on 10 μL each of the above mRNA samples with 2.5 μM oligo dT12 VA in a 20 μL reaction volume containing 40 units RNAse inhibitor and 200 units of Superscript II Reverse Transcriptase (Invitrogen Corporation, CA, USA). The reverse transcription reaction was followed according to Invitrogen’s protocol. Differential display PCR was performed in a 20 μL total volume per primer pair and 1 μL of the above cDNA, employing the Advantage cDNA PCR kit (Clontech, CA, USA). The PCR reaction conditions were 94ÖC-5 min, 40ÖC-5 min, 68ÖC-5 min for one cycle; 94ÖC-30 sec, 40ÖC-30 sec, 68ÖC-5 min for two cycles; 94ÖC-20 sec, 60ÖC-30 sec, 68ÖC-2 min for 23 cycles. The PCR reactions were performed in duplicate and the results were run on 1.6% (w/v) agarose gel (12 cm W ¥ 0.5 cm H ¥ 14 cm L) at 24 V for 16 h. Following this procedure, the gels were stained in ethidium bromide at 0.1 μg mL-1 (w/v), and the results were documented with Polaroid film. The desired fragments were excised from the gel (see below).


Design and choice of primers for differential display

Adapting the results of Consalez et al (1999), four arbitrary primers were chosen for their high efficiency in yielding large numbers of PCR products and for their selectivity for coding regions versus untranslated regions of transcripts. They are ID # 51 (TGCCGACTCTGC/G), ID # 163 (ACGTGCCCAGCA/T), ID #688 (AAGCTGCTCGCG/C), and ID # 942 (ACGCCATCGACC/G).


DNA extraction, cloning, and sequencing

Excised fragments were extracted according to QIAEX II protocol (Qiagen USA) and eluted in 8 μL of 10 mM Tris-HCl (pH 8.5). Cloning of the above fragments was performed with a TOPO-TA Cloning Kit (Invitogen, Carlsbad, CA). At least five colonies were selected and cultured overnight in LB liquid medium containing 50 μg mL-1 kanamycin. Plasmid isolation was performed using a SpinPreptm Plasmid Kit (Novagen, US). Plasmids were analyzed for inserts by restriction digestion followed by agarose gel electrophoresis. Sequencing of desired clones was performed by the Sequencing Core facility at the University of Florida using a Perkin Elmer/Applied Biosystems Model 373A and 377 sequencer.


Sequencing analysis

The nucleotide sequence data obtained was edited, so that only the unambiguous sequence information was included as query in the BLAST (Basic Local Alignment Search Tool) program available online at the NCBI (National Center for Biotechnology Information) web site (www.ncbi.nlm.nih.gov). The nucleotide sequences of each clone were queried against the protein database using blastx option in which query is converted into protein sequences in all six reading frames.

RESULTS AND DISCUSSION

Verification of C3 and C4 Plant Material

To establish the C3 or C4 status of the plant material, analyses of phosphoenolpyruvate carboxylase (PEPC) activity, the initial enzyme in the C4 cycle, were performed on the Hydrilla leaves. The specific activities were 22 and 25 μmol mg-1 protein for C3 light- and dark-harvested plant material, respectively. In contrast, C4 plant material showed over 20-fold greater specific activities for light- and dark-harvested material of 477 and 655 μmol mg-1 protein, respectively.


Differential Display

A differential display technique was used to identify up-regulated or unique genes in the C4- induced plant tissue relative to C3 tissue. Figure 1 shows C3 and C4 cDNA fragments resolved on a 1.6 % agarose gel after PCR with oligo dT12 VA and the four random primers listed above. One of the primers failed to amplify any products (Lanes 4 and 5) while primer #944 shown in Lanes 2 and 3 amplified many differentially expressed fragments. The rest of the primers produced very similar patterns of fragments.

Figure 1. Differential display pattern of C3 and C4 cDNA with four different arbitrary primers and a single oligo-dT anchor primer in a 1.6% agarose gel electrophoresed for 16 h.
Figure 1. Differential display pattern of C3 and C4 cDNA with four different arbitrary primers and a single oligo-dT anchor primer in a 1.6% agarose gel electrophoresed for 16 h. Lanes 2 and 3 show fragments produced from PCR with primer #944. Lanes 4 and 5 represent primer # 688 and did not produce any fragments. Lane 6 and 7 show fragments produced with primer #163 and Lanes 8 and 9 have fragments produced from primer #51. Lane 2 shows multiple genes that are up-regulated or unique to the C4 system and are represented with arrows. These bands were excised and subjected to PCR but failed to produce results. The fragments ranged in size from 1.0-2.0 Kb. 2-log and 100 bp molecular markers are shown on left and right side, respectively.


Figure 2 shows the reproducible results of a repeat PCR employing oligo dT12 VA with primer 944. The products were subjected to electrophoresis in duplicate lanes on 1.6% agarose gel under the conditions mentioned earlier. At least four unique or up-regulated bands were clearly visible in the lane where C4 cDNA was used as template. Their sizes ranged from 0.9 to 1.75 Kb. Multiple bands were excised from positions shown on the Figure.

Figure 2. Repeat differential display for reproducibility.
Figure 2. Repeat differential display for reproducibility. PCR product with primer #944 and anchored oligo-dT were separated on 1.6% agarose gel. Lanes with marker, C3 and C4 cDNA products are indicated. Arrows shown on the gel: fragments A (1.75Kb); B (1.5); C (1.3Kb); and S (0.9-1.1Kb). They were chosen as unique or up-regulated genes in the C4 system.

Figure 2. Repeat differential display for reproducibility.
Figure 2. Repeat differential display for reproducibility.E. coli was transformed with 2 μL ligation mix (gel extracted cDNA with pCR4-TOPO vector) and spread on four LB-agarose plates with kanamycin (marked A, B, C and S), and incubated overnight at 37ÖC. Lanes A1-A5, B1-B5, C1-C5 and S1-S5 represent five independent clones selected from the corresponding plates above and grown overnight in 5 mL of LB-liquid medium with kanamycin. Plasmid DNA was isolated from each of these clones, digested with 1 unit of restriction enzyme (Eco RI) overnight at 37ÖC. Arrows indicate the clones selected for sequencing.


Sequencing

Blast searches of nucleotide sequences from C4 fragments of unique or up- regulated genes showed homology with entries from the Arabidopsis thaliana genome (Table 1). Three showed similarity to a putative arginine methyltransferase. The methylation of arginine residues has been implicated in the regulation of signal transduction, transcription, RNA transport, and possibly splicing. Another nucleotide sequence showed approximately 60% homology with an early dehydration response protein (ERD4) from Arabidopsis thaliana. A further sequence showed homology with a hypothetical protein that is similar to a retro element from rice (Oryza sativa). Lastly, one matched a Ser/Thr kinase protein, which has interest to us as a regulatory protein for the PEPC enzyme.


Table 1
Results of BLAST Query
Each nucleotide sequence was tranlated into protein sequences in all six reading frames. The tranlated protein products were then compared against the NCBI protein databases
Clone Fragment length* (Kb) Forward sequence1 (bp) Reverse sequence2 (bp) Most related sequence E value3
A1 ∼2.0 663 705 gi|9454581|gb|AAF87904.1|AC01
5447_14 Similar to protein kinases [Arabisopsis thaliana]
Length=705		 9e-4
B1 ∼1.2 546 NA gi|17381028|gb|AAL36326.1|
putative arginine methyltransferase
pam 1 [Arabisopsis thaliana]
Length=390		 1e-51
B3 ∼1.4 496 605

gi|12322128|gb|AAG51102.1|AC0
25295_10 unknown protein
[Arabidopsis thaliana]
Length=724

 1e-45
B4 ∼2.0 723 NA gi|15375406|dbj|BAB63915.1|
ERD4 protein [Arabidopsis thaliana]
Length=640		 2e-76
C1 ∼1.4 575 NA gi|22711570|gb|AAN04521.1|
Hypothetical protein with similarity to putative retroelements [Oryza sativa (japonica cultivar-group)]
Length=237		 2e-09
C3 ∼1.2 724 NA gi|17381028|gb|AAL36326.1|
putative arginine methyltransferase pam 1 [Arabidopsis thaliana]
Length=390		 3e-65
S1 ∼1.5 536 642 gi|28564636|dbj|BAC57818.1|
PO577B11.15 [Oryza sativa(japonica cultivar-group)]
Length=1133		 6e-68
S2 ∼1.2 590 705 gi|23297369|gb|AAN12952.1|
arginine methyltransferase pam 1 [Arabidopsis thaliana]
Length=390		 6e-61

* fragment length is the length as found by restriction digestion in Fig. 3
1 forward sequence is the number of base-pair in the chromatogram marked and selected for blastx
2 reverse sequence - same as above
3 E value or the Expert value is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size

CONCLUSIONS

One of the interesting issues currently in crop biotechnology concerns the minimum components needed to transform a C3 crop, such as rice, to a more productive C4 system. The inducible Hydrilla system provides an excellent opportunity to study the minimum biochemical elements to operate a C4 photosynthetic system. Low [CO2] and high temperatures are two environmental parameters involved in the shift of Hydrilla from C3- to C4-photosynthesis. The unique facultative nature of this plant allows the simultaneous examination of the genes involved in both the C3 and C4 states and enables us to document the differences in their expression. In addition, the differential display used here is an excellent method for studying potentially up-regulated genes when the C4 system is induced, and differences in transcription between the two photosynthetic states.

The C4 plant material showed up-regulation of at least five genes visible by differential display. It is evident from the these results that a number of up-regulated or uniquely expressed components exist in addition to key enzymes such as PEPC. It is also encouraging that these results were obtained using only four primers, out of potentially 96 (Consalez et al, 1999). This suggests that differential display is a very appropriate method for mining more C4-specific components from this plant.

REFERENCES


Bowes G, Rao SK, Estavillo GM, Reiskind JB (2002) C4 mechanisms in aquatic angiosperms: comparison with terrestrial C4 systems. Funct Plant Biol 29: 379-392

Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254

Consalez GG, Cabibbo A, Corradi A, Alli C, Sardella M, Sitia R, Fesce R (1999) A computer-driven approach to PCR-based differential screening, alternative to differential display. Bioinformatics 15: 93-105

Liang P, Pardee A (1992) Diffential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971

Magnin NC, Cooley BA, Reiskind JB, Bowes G (1997) Regulation and localization of key enzymes during the induction of Kranz-less, C4-type photosynthesis in Hydrilla verticillata. Plant Physiol 115: 1681-1689

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Rao SK, Magnin NC, Reiskind JB, Bowes G (2002) Photosynthetic and other phosphoenolpyruvate carboxlase isoforms in the single-cell, facultative C4 system of Hydrilla verticillata. Plant Physiol 130: 876-886

Reiskind JB, Madsen TV, Van Ginkel LC, Bowes G (1997) Evidence that inducible C4-type photosynthesis is a chloroplastic CO2 concentrating mechanism in Hydrilla, a submersed monocot. Plant Cell Environ 20: 211-220.

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