Transcriptional analysis of short- term boron deficiency in Arabidopsis indicates altered sulphur metabolism
Alves M1, Saibo NJM1, Gonçalves D1,2, Ricardo CP1,*
1
Instituto de Tecnologia Química e Biológica, Av. República, Estação Agronómica Nacional, 2780- 157 Oeiras, Portugal.
2
Instituto Superior Técnico Av. Rovisco Pais, 1049- 0 01 Lisboa, Portugal.
* Corresponding author: [email protected]
CONTRIBUTION: The first author performed all the experimental work and writing with the exception of the GeneChip hybridization, GeneChip data analysis and boron quantification.
KEYWORDS: Arabidopsis thaliana, Boron deficiency, Calcium
SUMMARY
For more than 80 years, boron ( B) has been recognized as an essential microelement for higher plants, but in spite of this its precise role remains elusive. In order to gain insight into the participation of B in the metabolism of higher plants, we have analysed Arabidopsis thaliana for transcriptional alterations caused by short- term B deficiency. Given that several studies suggest an interaction between B and calcium ( Ca) in plants, the transcriptional profile of A. thaliana plants under Ca- deficiency was also analysed. The microarray analysis revealed that the expression of 102 genes was altered 2 days after withholding B and 208 by day 4. For the Ca deficiency, 2 days after Ca withholding morphological symptoms were already evident and more than 2,500 genes were altered, and at 4 days the plants showed necrotic symptoms and only 270 genes were differentially expressed. Of the 298 B- responsive genes, 37 were identified has having the same expression trend at 2 and 4 days of deficiency ( 15 up- regulated and 22 down- regulated) and, so, were selected for further analysis. Thirteen genes that had a fold- change higher than 2 were chosen for RT- qPCR analysis and since we were able to validate all of them we included all the 37 genes for the detailed discussion. In this set, we found genes related with cell wall biosynthesis ( 24%) , sulphur metabolism ( 24%) , transcriptional factors and hormones
( 18%) , while genes related with miscellaneous and unknown functions represent 13% and 16%, respectively. Altered cell wall biosynthesis is a process already described for B- deficient plants, whereas altered sulphur metabolism due to B deficiency is a rather novel observation.
INTRODUCTION
Boron ( B) is known to be an essential micronutrient for higher plants since 1923 ( Warington 1923) , but only in the 90’ s it was reported to participate in the cell wall structure as a cross linker of the cell wall pectin, rhamnogalacturonan- II ( RG- II) ( Kobayashi et al. 1996, O’ Neill et al. 1996, 2001) . However, this participation does not seem enough to explain all the symptoms of B deficiency in plants.
There are symptoms of short- term B deficiency that remain unexplained, such as the quantitative and qualitative changes observed in the phenolic metabolism of tobacco ( Camacho- Cristóbal et al. 2002) and the increased levels of cytoskeletal proteins in both Arabidopsis and maize roots ( Yu et al. 2001, 2003) . The enhancement of cytoskeleton protein levels and the altered polymerization patterns were hypothesized to be involved in the mechanical reinforcement of the cell periphery complex. Another
observed symptom that results from short- term B deprivation is the inhibition of pectin endocytosis from the cell walls of meristematic cells in maize and wheat root apices, which could be related to endocytosis- mediated pectin signalling ( Yu et al. 2002) . Additionally, it has been suggested that the metabolic functions of calcium ( Ca) and B in plants may be inter- linked. For instance, in nodulation processes some effects of B deficiency were ameliorated by Ca addition ( Redondo- Nieto et al. 2003, Koshiba et al. 2010) , and genetic studies showed that the expression of some genes affected by B deficiency could be reversed by Ca supplementation ( Bolaños et al. 2004) . Stability studies of different B fractions suggested that B cross- link of RG- II may result in conformational changes that can create binding sites for Ca ions, which increase pectin association and rigidity ( Kobayashi et al. 1999) . Since little is known about the roles of B in higher plants, and given the possible interactions between B and Ca, we have analysed the transcriptional profile of Arabidopsis thaliana plants subjected to 2 and 4 days of B or Ca deficiency. Our results provide insight into the early responses to B deprivation in Arabidopsis.
MATERIALS AND METHODS
Plant material and growth conditions
Arabidopsis thaliana cv. Columbia seeds were sown in 1L plastic
containers filled with perlite, which has a low B content ( Alves et al. 2006) . After a period of 2 days at 4ºC, the containers were transferred to a growth chamber ( Fitoclima 700 EDT4, Aralab) at a temperature of 16/ 22ºC ( night/ day) , with a 16h photoperiod and a light intensity of 150
µ
mol m- 2 s- 1, PAR. The plants were watered every other day with a complete nutrient solution ( Arnon 1938) in which the B concentration was 25µM
and the Ca concentration 3mM. Boron or Ca deficiencies were imposed 17 days after sowing by daily watering the plants with a B- free nutrient solution or with a Ca- free nutrient solution corrected for the nitrate content with magnesium and potassium salts.The Arabidopsis plants were harvested 2 and 4 days after B or Ca suppression and kept frozen at - 80ºC until further use. The Arabidopsis plants grown for longer than 12 days under B or Ca deficiency were only used for morphological analysis. For dry weight determinations, the plants were oven- dried for 48h at 80ºC.
Boron quantification
Boron was quantified following the Azomethine- H method ( Sungur and Okur 2009) adapted for micro- assays. The plants were ashed
overnight in an oven at 550ºC and the ashes dissolved in 3N HCl. The solution was filtered through a 0.45
µ
m filter and 20µ
L were used for analysis. To each well were added20µL of water, 80µL
of azomethine- H solution [ 0.45% ( w/ v) azometinhe- H and 2% ( w/ v) ascorbic acid] , and 80µL of
buffer solution at pH 5.7 [ 48% ( w/ v) ammonium acetate, 3% ( w/ v) EDTA disodium salt and 24% ( v/ v) acetic acid] . The mixture was kept at room temperature for 40min. The absorbance was measured in a UV/ Vis 96- well plate reader ( Powerwave xs, Bioteck) at 420nm. A calibration curve was prepared using different concentrations of aqueous B solutions. All analyses were performed in triplicate.RNA Isolation, Target Synthesis and Hybridization to Affymetrix GeneChips
Total RNA was extracted using the RNeasy Plant Mini Kit ( Qiagen, Hilden, Germany) . Concentration and purity were determined by spectrophotometry and integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay ( Agilent Technologies, Palo Alto, CA) . The RNA was processed for use on Affymetrix ( Santa Clara, CA, USA) Arabidopsis ATH1 Genome Arrays, according to the manufacturer’ s One- Cycle Target Labelling Assay.
Briefly, 5µg of total
RNA spiked with Poly- A RNA controlsAffymetrix) was used in a reverse transcription reaction ( One- Cycle DNA synthesis kit; Affymetrix) to generate first- strand cDNA. After second- strand synthesis, double- stranded cDNA was used in an in
vitro transcription ( IVT) reaction to generate biotinylated cRNA
( GeneChip Expression 3’ - Amplification Reagents for IVT- Labeling; Affymetrix) . Size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a