Abstract
The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here we report the generation of over 14,000 amiRNA-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol is developed here for isolating robust and reproducible amiRNA-mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA insensitive amiRNA line is isolated, which targets three genes encoding avirulence-induced gene2-like (AIG2) genes. A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led here to isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 14,000 T2 amiRNA lines generated here provides a new platform for forward genetic screens and is being made available to the Arabidopsis Biological Resource Center (ABRC) and optimized procedures for amiRNA screening and controls are described.
Highlight The generation of over 14,000 amiRNA-expressing plants is reported that are being made publicly available enabling screens of redundant genes in Arabidopsis. Identification of known and new genes is reported.
Introduction
The presence of large gene families in plants including Arabidopsis thaliana (Arabidopsis Genome Initiative, 2000) leads to functional genetic redundancies or partial functional overlap among closely related genes. Functional overlap and partial or complete redundancy between different family members has been proposed to provide a buffer for loss or gain of function mutation events and mechanistic robustness of cellular networks (Wagner, 2005). This is considered to be a main reason for the lack of observable phenotypes in single-gene deletion mutants and increasing severity of phenotypes in higher order mutants of homologous genes (Ma et al., 2009; Park et al., 2009). Identification and characterization of functionally overlapping genes in genetic screens is limited, as is evident by the relatively low number (591 of all Arabidopsis genes) of genes not associated with a single mutant phenotype (Lloyd and Meinke, 2012). Analysis of genome-wide gene family definitions showed that the Arabidopsis genome includes over 22,000 genes belonging to gene families (Hauser et al., 2013). Strategies and tools have been developed to enable screens of the functionally redundant gene space. Recently, an artificial microRNA (amiRNA) based computational design approach was introduced (Hauser et al., 2013). AmiRNAs designed to specifically target diverse combinations of gene family members or combinations of subfamily members enable the screening of partial over-lapping homologous gene functions at a genome-wide scale. The presented platform also provides an approach for capture of homologous gene silencing phenotypes, for which higher order loss of function mutants would lead to lethality, as illustrated by a mutant identified here.
Here we report the generation of over 14,000 T2 amiRNA lines by transformation of A. thaliana Col-0 with a previously published amiRNA library (Hauser et al., 2013) and screening of T2 amiRNA lines for abscisic acid (ABA) insensitive seed germination phenotypes or plants with low CO2 insensitive high leaf temperature phenotypes. Methods are described to identify robust amiRNA mutants and how to avoid pitfalls of this approach. The screen rapidly identified two amiRNAs which target three PYR/RCAR ABA receptors (Ma et al., 2009; Park et al., 2009) or six SNF1-related kinase (SnRK2s (Mustilli, 2002; Yoshida et al., 2002; Fujii and Zhu, 2009)) encoding genes known to be involved in ABA-mediated control of seed germination. One candidate line which shows an ABA insensitive seed germination phenotype contains an amiRNA that targets three genes of unknown function which are annotated as AIG2A (AT3G28930), AIG2LA (AT5G39720) and AIG2LB (AT5G39730). One amiRNA which causes a low CO2 insensitive high leaf temperature phenotype targets two genes encoding proteasomal !2-subunits annotated as PAB1 (AT1G16470) and PAB2 (AT1G79210), for which double mutation causes lethality. New amiRNA lines that target the proteasomal α([0-9]+)-subunit annotated as PAG1 (AT2G27027), was constructed resulting in a similar stomatal phenotype. Together these observations indicate a rate-limiting role of the intact proteasome for stomatal opening responses.
Materials and Methods
Plant material, growth conditions and transformation
Arabidopsis thaliana accession Columbia-0 was used as the background for all amiRNA transformations of the library. Surface sterilized seeds (15 min 70% Ethanol, 0.1% Sodium dodecyl sulfate; 3-4 washes with ∼ 100% Ethanol; alternatively 10 min 50 % bleach, 0.05% Tween 20; 4 - 6 washes with water (Lindsey et al., 2017) of A. thaliana were cold-treated for 2 - 5 days at 4°C and germinated on half strength Murashige and Skoog basal medium supplemented with Gamborg’s vitamins (Sigma-Aldrich (Murashige and Skoog, 1962; Gamborg et al., 1968), 0.8% Phytoagar (Difco, Franklin Lakes, NJ) and pH adjusted (pH 5.8; 4-Morpholinoethane sulfonic acid (2.6 mM; Sigma-Aldrich) titrated with potassium hydroxide). After 5 – 7 days, plants were transferred to plastic pots containing sterilized premixed soil (Sunshine Professional Blend LC1 RS; Sunshine; supplemented with an appropriate amount of insecticide (Marathon, Gnatrol)) and propagated under the following conditions: long day (16hr light/ 8 hrs. dark); 23-27 °C; 20-70% humidity, 60-100 mmol m−2 sec−1 light.
Plant transformation by floral dip was performed as described elsewhere (Clough and Bent, 1998) with the following modifications: Agrobacterium tumefaciens GV3101::pMP90 (Koncz and Schell, 1986) was grown under selection of all markers, i.e. genomic (rifampicin), Ti-plasmid (gentamicin), pSOUP (tetracycline) and T-DNA plasmid (spectinomycin). The infiltration medium for resuspension of the bacteria and floral dip contained 5% Sucrose (w/v) and 0.02% (v/v) Silwet L-77 (Clough and Bent, 1998).
Large scale transformation with the amiRNA library pools (Hauser et al., 2013) was performed as described elsewhere (Cutler et al., 2000) with the following modifications. One microgram DNA of each amiRNA sub library (Hauser et al., 2013) was electroporated into a total of 500 μl electrocompetent A. tumefaciens cells. The 20 bp and 21 bp amiRNA sub library variants for each pool were individually electroporated and combined at this stage. After two hours at 30 °C in non-selective Luria Bertani Miller medium (LB, Teknova) the cells were spread on 20 LB plates (1.5% agar; 150 mm diameter) containing all the appropriate antibiotics (rifampicin, gentamycin, tetracycline, spectinomycin) and grown for three days at 30°C. The bacteria were scraped from the plates, re-suspended in 5 ml LB and concentrated by centrifugation for 20 min at 5855 x g. Plants were transformed by spraying the flowers with this suspension of the bacteria in infiltration medium (adjusted to an optical density at 600 nm of 0.5) twice with one week between the treatments. T1 plants were selected on plates supplemented with 75 μM phosphinotricin or directly on soil by spraying diluted herbicide (1000 x dilution, Finale®; Bayer, North Carolina Research Triangle Park, NC) four times with 2 - 7 days between the treatment. Herbicide resistant plants were transferred to soil and grown to full maturity and T2 seeds collected from individual plants. When appropriate, media for growth of bacteria or plant selection contained the following concentrations of antibiotics (mg ml−1): carbenicillin 100, gentamycin 25, kanamycin 30, rifampicin 50, spectinomycin 100, tetracycline 10, and phosphinotricin 15.
Screen for abscisic acid insensitive seed germination phenotype
T2 plants were screened individually for insensitivity of seed germination to abscisic acid in 96 well plates (100 μl 0.1 % Agarose supplemented with 2 μM (±)-ABA, Sigma-Aldrich). Approximately 10 - 20 seeds were used from each T2 plant. For the pooled screening, approximately 10 - 50 seeds of 90 individual T2 plants were mixed, surface sterilized and sprinkled onto agar plates (3 μM (±)-ABA; Sigma-Aldrich). As control for ABA insensitivity, abi4-1 (ABRC, CS8104) or abi5-1 (ABRC, CS8105) were used, Col-0 was used as wild-type control. A putative abscisic acid (ABA) insensitive phenotype was scored in a binary manner for similarity to the abi4-1 or abi5-1 phenotype and difference to wild type after 5 - 8 days using green cotyledons as indicator (Kuhn et al., 2006). For lines which showed a putative ABA insensitivity, the seed germination assay was repeated by propagating individual T2 seedlings to the next generation (T3) and using seeds of the T3 generation for ABA sensitivity assays. This time, seeds were placed on plates with and without ABA (2 μM (±)-ABA; Sigma-Aldrich) and images were taken daily for 7 days and emergence of radicles and cotyledons was counted manually using Fiji (Schindelin et al., 2012). For candidates of the individual screen the T2 seeds were used for the repetition of the germination assay.
For candidates of the pooled screen ABA insensitive seedlings were transferred to plates containing 75 μM phosphinotricin and after 7 – 10 days resistant seedlings were transferred to soil, grown up to full maturity and the T3 seeds used for the validation of the ABA insensitive germination phenotype.
Screen for CO2 insensitive leaf temperature phenotype
Seeds of T2 plants were germinated in 96 pots flats (254 mm x 508 mm; East Jordan Plastics, East Jordan, MI) on soil with each pot containing seeds from one plant. After seven days seedlings were sprayed with a 1000 x dilution of Finale® (Bayer, Bayer, North Carolina Research Triangle Park, NC) and two to three days later pale seedlings were removed and only one healthy dark green seedling was left per pot. After 19 days under standard growth conditions the plants were exposed to 150 ppm CO2 for two hours in a Percival growth chamber. A first set of thermal images was taken with a FLIR A320 thermal imaging camera (FLIR, Wilsonville, OR). Subsequently the plants were exposed to ≥ 800 ppm CO2 and after two hours a second set of thermal images was taken. Control plants included in the experiments were ht1-2 (Hashimoto et al., 2006), ost1-3 and wild-type Col-0. Thermal images were converted into Flexible Image Transport System format (fits) using the ExaminIR software (FLIR, Wilsonville, OR). For the screen using the 96-pot flat format, the temperature of plant leaves and the surrounding soil were measured using Fiji (Schindelin et al., 2012). The soil temperature served as location specific reference to compensate for temperature variation depending on the position in the 96 pots flat. Either the temperature difference between plant leaves and surrounding soil or the average temperature of plant leaves were used as a quantitative measure. Plants with more than one degree Celsius difference to soil were considered as primary candidates and subject to further testing. The high-temperature leaf phenotype of ht1-2 was used as a reference for CO2 insensitivity. To test the reproducibility of the CO2-dependent leaf temperature phenotype of putative candidates, T2 plants were grown in triplicate and assayed again alongside with ht1-2 and wild-type control plants.
Identification of amiRNA sequences and testing reproducibility
Genomic DNA from candidates with a robust and reproducible phenotype was isolated as described elsewhere (Edwards et al., 1991) and the sequence of the amiRNA present was determined by sequencing of the PCR product (primers pha2804f and pha3479r; see Supplemental Table 1). Using the Target Search function available on the WMD3 website (Ossowski et al., 2008) putative amiRNA target genes were predicted. For independent confirmation of the phenotype independent lines were generated by cloning the identified amiRNA into pFH0032 (Supplemental Table 2; (Hauser et al., 2013) and transforming it into Arabidopsis Col-0. Confirmed phenotypes were further analyzed by using single knock out mutants, higher order mutants generated by crossing and/or generating amiRNAs which target subsets of the initial target genes (Supplemental Table 3).
Gas exchange analyses
Stomatal conductance of H2O (gs) was measured in leaves of 5 to 6-week-old plants using portable gas exchange systems (LI-6400 and LI-6800, LI-COR, Lincoln, Nebraska), starting 2 hours after growth chamber light onset. For intact single leaf ABA treatments, a LED light source set at 150 µmol m−2 s−1 (10% blue) and a chamber temperature of 21 °C was used. Leaves were equilibrated for one hour at a relative humidity of 70-72%, airflow of 200 rpm and CO2 concentration of 400 ppm. After one-hour, steady-state stomatal conductance was recorded ten minutes before the addition of ABA to the petioles in water at the indicated concentration. For light-response measurements, plants were kept in the dark for 18 hours prior to experiments. Stomatal conductance of a single intact leaf in the dark was recorded for 10 min, followed by red light treatment of 600 μmol m−2 s−1. After 20 minutes of red light treatment, additional blue light was applied at 10 μmol m−2 s−1. The incoming air humidity was kept at 62-65% and air flow at 200 rpm. For stomatal conductance measurements of single intact leaf CO2-responses, incoming relative air humidity was kept at 62-65% and the imposed changes in CO2 concentration were applied as indicated. Leaves were attached to intact plants and were equilibrated for one hour before the measurements. The data presented represent n ≥ three leaves with each leaf from independent plants per genotype per treatment.
qRT-PCR analysis
Total RNA (500ng) was reverse transcribed using the first-strand cDNA synthesis kit (GE Healthcare). qRT-PCR analyses were performed using 3-fold-diluted cDNA, Maxima SYBR Green Rox/qPCR Master Mix (Thermo Scientific). The housekeeping PDF2 gene was used as an internal control. The threshold cycle (CT) was determined by the instrument, and the Δ ΔCT method was used to calculate the fold change in each gene (Livak and Schmittgen, 2001). For RAB18 gene expression measurements, two-week-old seedlings were treated with ABA for nine hours, final concentration of 20 μM and total RNA extraction was used.
Results and Discussion
Generation of amiRNA library plants
We have previously described the generation of an amiRNA library consisting of ten sub libraries that represent 22,000 individual amiRNA designs (Hauser et al., 2013). Deep sequencing of these 10 sub libraries showed that Δ 95% of the designed amiRNAs were present in these sub libraries (Hauser et al., 2013). The amiRNA library was transformed first into Agrobacterium tumefaciens and then into Arabidopsis Col-0. Over a period of over four years, the amiRNA library consisting of ten sub libraries was transformed and T1 seeds harvested. Using plate or soil-based selection methods, herbicide resistant T1 plants were grown and T2 seeds from over 14,000 individual plants were harvested (Table 1). The transformation rate varied over a range from 0.08% to 0.76% with an average of 0.25%. During the course of this research, approximately 3,000 additional T2 lines were generated expressing amiRNAs that target homologous transporter-encoding gene family members. These 3,000 lines will also be made available to the ABRC, such that over 14,000 total T2 lines will be submitted for use by the community.
Screen for ABA insensitive seed germination phenotype
In total over 2,500 T2 amiRNA lines were screened individually and over 5,000 T2 amiRNA lines were screened in pools for ABA insensitive germination phenotypes (Figure 1). In the primary screen using individual plants in a 96-well plate format, 59 putative candidates were identified. In the primary screen using pools of 90 plants with 25 - 80 seeds per line, 340 putative candidates representing an unknown number of lines were identified (Figure 1).
These candidates were subjected to further analysis in a secondary screen (Figure 2). The cotyledon emergence phenotype of 24 T3 seedlings from a total of 76 retested plants showed a more reduced ABA sensitivity that was clearly different from wild type and less severe than the abi4-1 and abi5-1 controls (Figure 2A). From the 59 putative candidates identified using the individual screening approach, the amiRNA line p8l1257 showed a reproducible partial insensitivity to ABA in the T3 generation (Figure 3). Only the amiRNA in candidates with the most robust phenotypes were determined by sequencing. Two of the amiRNA-targeted gene sets identified in 24 seedlings with reproducible phenotypes are known core components of the ABA signal transduction cascade (Figure 2, Table 2). These include amiRNA lines that target the three ABA receptors PYR1 (RCAR11), PYL4 (RCAR10) and PYL6 (RCAR9) (Figure 2B, C and Table 2). Furthermore, amiRNA-expressing plants that target six members of the SnRK2 protein kinase family (Mustilli, 2002; Yoshida et al., 2002; Fujii and Zhu, 2009) were isolated in this screen, including the three SnRK2 protein kinases, SnRK2.2, SnRK2.3 and SnRK2.6 (OST1) that are known to be required for abscisic acid signaling (Figure 2B, C and Table 2; (Mustilli, 2002; Yoshida et al., 2002; Fujii and Zhu, 2009)).
Notably, Figure 2D shows a strong variation in the cotyledon emergence phenotype among plants expressing the same amiRNA that targets six SnRK2 kinase transcripts. This variation might be responsible for the high number of variable candidates which did not show a robust phenotype following the primary screen. Additional amiRNA lines were isolated as putative mutants and the amiRNA sequence was determined (Supplemental Table 4). Although some of the predicted targets might be expected to affect abscisic acid responses, rescreening of these putative mutants did not show consistently robust reproducible phenotypes. Thus, amiRNAs appear to produce phenotypes that may be variable even within the same line. These findings led us to develop a protocol in which:
Only putative mutants that showed a consistent phenotype when screening seeds from the next generation of plants were selected.
Only lines that showed similar phenotypes upon re-transformation with new amiRNAs that are predicted to target the same genes were selected. Furthermore, based on the variation observed here in the secondary screen it is advisable to investigate over ten independent transformed lines (Schwab et al., 2006; Hauser et al., 2013) in the future to determine which amiRNAs produce phenotypes that can be carried forward. The isolation of amiRNA lines targeting functionally over-lapping PYR/RCAR ABA receptor and SnRK2 protein kinase genes, that would not be isolated in traditional forward genetic mutant screens, provides a proof of principle that functionally redundant genes can be isolated in forward genetic screening using this new amiRNA resource. The inclusion of control lines and the validation steps described above should enable screening for diverse phenotypes using the lines generated here that are being provided to ABRC.
AmiRNA lines targeting three avirulence-induced genes (AIGs) show partial insensitivity to ABA-inhibition of seed germination but not to ABA-induced stomatal closure
The amiRNA in line p8l1257 isolated in the present screen targets a new set of three genes (Figure 3A and 3B). Previous research annotated these genes based on their mRNA upregulation in a transcriptomic study after infection with Pseudomonas syringae pv maculicola carrying avrRpt2 (avrRpt2-induced gene, AIG2) (Reuber and Ausubel, 1996). However, these genes have not been previously described to be involved in ABA-mediated control of seed germination or other phenotypic responses in plants.
The line p8l1257 was named amiRNA-AIG here and was further tested by analyzing seed germination with additional T2 generation seeds from the original p8l1257 stock (Figure 3). Germination properties were compared to a control amiRNA line targeting the human myosin 2 (amiRNA-HsMYO), that has no targeted genes in Arabidopsis (Hauser et al., 2013). After 12 days on plates containing 2 μM ABA, the amiRNA-AIG line showed cotyledon greening in contrast to the control amiRNA-HsMYO line (Figure 3A). The effect of the amiRNA-AIG on the expression of a known ABA induced gene RAB18 was analyzed by qRT-PCR (Figure S1). The ABA mediated induction of RAB18 expression was substantially reduced in the amiRNA-AIG line indicating a role of the targeted AIG genes in ABA signal transduction.
Since two out of the three genes are tandemly repeated, generation of double mutants using T-DNA insertion knockouts would be limited. Therefore, five new amiRNA lines were generated which target subsets of genes targeted by the original amiRNA-AIG to verify the relevance of the predicted AIG target genes. AmiRNAs 1, 2 and 3 targeted each a single AIG (Figure 3B; Supplemental Table 3). AmiRNA 4 targeted two tandem-repeat AIG genes and amiRNA 5 targeted all three AIG genes targeted in the original amiRNA-AIG line, but with a different amiRNA sequence (Figure 3B, see Supplemental Table 3 for amiRNA sequences). When the T2 seeds expressing these five new amiRNAs were tested in a seed germination assay with 0.5 μM ABA, only the amiRNA 4 and amiRNA 5-expressing lines showed less sensitivity to ABA compared to the control amiRNA-HsMYO line in cotyledon greening (Figure 3C). The expression of all three putative target genes (AT5G39720, AT5G39730, AT3G28930) was analyzed using qRT-PCR in the originally isolated amiRNA-AIG line and in all the amiRNA lines 1 to 5 (Figure S2). The amiRNA efficiency of transcriptional inhibition varies between the lines, target transcript(s) and amiRNA sequence. Note that microRNA silencing in plants occurs via two mechanisms, (a) the degradation of transcripts and (b) inhibition of translation (Brodersen et al., 2008). Thus, quantification of targeted transcripts may not fully show the degree of silencing of target genes. Combined, these data provide evidence that the original amiRNA-AIG phenotype is attributable to silencing of more than one AIG gene, suggesting overlapping homologous gene functions.
The original amiRNA-AIG line was also investigated to determine if it affects ABA-induced stomatal closure using an intact leaf gas exchange analysis approach. When ABA was applied to the transpiration stream of intact leaves at a final concentration of 2 μM, both the control amiRNA-HsMYO line and the amiRNA-AIG lines showed an ABA-induced decrease in stomatal conductance to H2O (gs, Figure 4A). Normalization of the stomatal conductance data showed no dramatic difference in ABA-induced stomatal closure between amiRNA-HsMYO and amiRNA-AIG (Figure 4B). Together, the present data show that the isolated amiRNA-AIG line is less sensitive to ABA-inhibition of seed germination.
The AIG2 genes are functionally annotated as putative γ-glutamyl cyclotransferases (GGCTs, EC:4.3.2.9) based on their similarity to the human orthologue (HsGGCT; O75223). AIG2LA and AIG2LB share only 16% and 17% identity respectively to the human orthologue. GGCTs have been described to cleave γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline (Oakley et al., 2008). Further research will be required to determine the mechanism by which AIG2s affect ABA inhibition of seed germination.
Screen for CO2 insensitive leaf temperature phenotype
In total, over 2,500 T2 amiRNA lines were screened individually for an altered leaf temperature response to a low CO2 concentration (150 ppm) by infra-red thermal imaging (Figure 5). Leaf temperature depends on various parameters including radiation absorption, air temperature and humidity (Merlot et al., 2002). Low ambient CO2 concentration leads to stomatal opening in Arabidopsis, causing an increased transpiration rate and thus a decrease in leaf temperature compared to the surrounding air. Mutants impaired in CO2-induced stomatal opening appear warmer compared to wild type plants. In the screen, we used the soil temperature as reference to compensate for the local temperature differences due to various factors including humidity of the soil. Wild-type plants and plants of the HIGH TEMPERATURE1-2 (ht1-2) mutant (Hashimoto et al., 2006) were included in all trays as controls. Based on visual inspection of the thermal images, plants with relatively higher leaf temperature under low [CO2] compared to the other plants in the same image were selected and the difference between the average leaf temperature and the surrounding soil was determined. The difference between leaf temperature and soil temperature was determined as reference for overall temperature and to compensate for local temperature differences. A set of 106 plants with more than one degree difference between the leaf temperature and the surrounding soil was defined as initial putative candidates for further testing (see Methods for details). For the rescreening of putative mutants, we set a high threshold for temperature differences in the selection of mutants compared to the wild-type strain of 1°C. The constitutive CO2 response mutant ht1-2, when exposed to low [CO2], shows a delta temperature above 1 °C between leaf and soil. Rescreening of these candidates in the T2 generation revealed an amiRNA line (p9l22) with a robust and reproducible impaired response to low CO2 (Figure 5B).
After exposure to low [CO2], the leaf temperature of the p9l22 line was compared to wild type (Col-0) and to the constitutive high CO2-response mutant ht1-2 (Figure 6A; (Hashimoto et al., 2006)). The leaves of the p9l22 line had a higher temperature than wild-type leaves and a similar temperature to ht1-2 leaves (Figure 6A). Stomatal index (SI) and density (SD) were calculated for wild type, the control amiRNA-HsMYO and p9l22 lines. No noteworthy differences were found between the genotypes (Figure S3; amiRNA-HsMYO vs. p9l22 line, One-Way ANOVA, p-value >0.05 for SI and SD).
To measure [CO2] responses in a time-resolved fashion, we measured stomatal conductance (gs) using a gas exchange analyzer. In the amiRNA-HsMYO control line, the shift from ambient (400 ppm) to low (150 ppm) [CO2] led to a rapid increase in stomatal conductance (Figure 6B). AmiRNA line p9l22 responded to the same treatment with a lower magnitude of stomatal opening (Figure 6B). Both lines showed stomatal closure in response to high (800 ppm) [CO2] exposure at similar rates (Figure 6B). To test whether line p9l22 is defective in response to other stimuli that cause stomatal opening, light-induced gs responses were investigated (Figure 6C). The control amiRNA-HsMYO and p9l22 lines were kept in the dark for 18 hours prior to the experiments and steady-state gs was measured. When red light (at 600 μmol m−2 s−1) was applied, the p9l22 line showed a reduced rate of gs increase when compared to the control line. The same was observed when blue light (at 10 μmol m−2 s−1) was superimposed on the red light background (Figure 6C). Thus, the amiRNA line p9l22 causes reduced responses to low CO2 concentration, red light and blue light.
The amiRNA in the p9l22 line was sequenced and is predicted to target two closely homologous proteasomal subunit genes (PAB1, At1G16470 and PAB2, At1G79210). Both PAB1 and PAB2 genes are the sole two genes that encode the 20S proteasome alpha 2 (α([0-9]+)) subunit (Baumeister et al., 1998). First, we attempted to isolate a double mutant (pab1 pab2) using T-DNA insertion lines (SALK_099950 and SALK_144987) (Alonso et al., 2003). After genotyping over 100 plants in the F2 generation, no homozygous double mutant was recovered. We concluded that the double mutation is very likely to be lethal.
Alternatively, a new amiRNA sequence targeting solely the PAB1 gene was cloned and transformed into the pab2-1 single mutant (SALK_144987). This new amiRNA line, pab2-1mut pab1amiRNA, was investigated in stomatal conductance analyses of [CO2] responses (Figure 7). Leaves were first exposed to high (900 ppm) [CO2] for one hour and steady-state gs was recorded. Shifts to low (150 ppm) [CO2] led to an increase in gs in both the pab2-1mut pab1amiRNA line and the control HsMYO-amiRNA line (Figure 7A). The normalized stomatal conductance data show that pab2mut pab1amiRNA line responds to low [CO2] with a reduced magnitude compared to the control line (Figure 7B).
Initial experiments were pursued to determine if modifications in the alpha ring of the 20S proteasome might be linked to the above phenotypes, or whether this mutation is specific to only !2 subunit mutations of the proteasome. The alpha ring of the 20S proteasome is composed of seven alpha subunits (Kurepa and Smalle, 2008). The p9l22 amiRNA targets the only two genes that encode the !2 subunit of the proteasome (Figure 7, inset highlighted in red). To determine whether other alpha subunits also affect the response to low [CO2], a second amiRNA line was generated, which targets the PAG1 gene (α([0-9]+) subunit, inset in Figure 7 highlighted in green), named pag1amiRNA. The α([0-9]+) subunit is encoded by a single gene in Arabidopsis (Kurepa and Smalle, 2008). When a pag1amiRNA line was tested in gs responses to [CO2] shifts, it showed a lower rate of stomatal opening when compared to the control amiRNA-HsMYO line (Figure 7C, D; One-Way ANOVA p-value >0.05). The expression levels of PAB1, PAB2 and PAG1 were analyzed in the p9l22 amiRNA line and also in pab2-1mut pab1amiRNA and pag1amiRNA lines using qRT-PCR (Figure S4). With the exception of the severely reduced PAB2 expression in the pab2-1mut pab1amiRNA line when compared to control lines, no clear evidence for knock down at the transcriptional level could be detected in the amiRNA lines, which may point to amiRNA mediated inhibition of translation (Brodersen et al., 2008).
The present findings show that the p9l22 amiRNA line is also partially impaired in red light-induced stomatal opening. Red light mediates stomatal opening in part via activation of photosynthesis and the resulting drop in internal concentration of CO2 (Ci) (Roelfsema et al., 2002; Matrosova et al., 2015). In addition, the p9l22 line is also partially impaired in blue light-induced stomatal opening. This suggests that a general regulator of stomatal opening is impaired in this line. As the proteasome mediates the degradation of proteins and reduced functions of α-ring subunits are predicted to increase protein levels, it is tempting to speculate that the phenotype observed might be correlated with an increased abundance of a negative regulator of stomatal opening. Further research will be required to test this or other hypotheses. In other studies, the 26S α([0-9]+) subunit, when overexpressed, enhanced thermotolerance and adaptation in rice and Arabidopsis, suggesting that proteasomal subunits can have rate-limiting roles in regulating plant physiological responses (Li et al., 2015).
Summary and Future Use
A library of over 14,000 total T2 generation amiRNA lines was created as a new resource to screen the redundant gene space in Arabidopsis. These amiRNA-expressing lines are being provided as individual lines to the Arabidopsis Biological Resource Center (ABRC). Given the observations and findings in the present study, lines will be available for high-throughput screening in pools of 90 lines per pool with approximately 25 to 50 seeds per individual amiRNA line in each pool. In each pool, the pooled seeds for screening will originate from one of the 10 sub-libraries that target gene family members with defined functional annotations (Table 1; (Hauser et al., 2013)). This approach will increase the probability of identifying interesting putative mutants in future screens despite the biological variability in amiRNA silencing lines found here (Figure 2D).
The screen for abscisic acid insensitive seed germination phenotypes identified two amiRNAs targeting PYR/RCAR ABA receptor genes and SnRK2 genes, that are both known groups of redundant key genes and proteins required for ABA signal transduction (Ma et al., 2009; Park et al., 2009). Isolating amiRNA lines in these known components serves as proof of principle for our approach. Moreover, screening this amiRNA population enables the identification of mutants that require co-silencing of homologous gene family members, which are less likely to be found in forward genetic screens of EMS or T-DNA mutagenized seed populations. Overall the presented amiRNA screen shows that amiRNA lines are prone to show a high rate of variable candidates with weak or non-robust phenotypes. Nevertheless, as shown here new mutants can be isolated. Furthermore, during the course of this research, this amiRNA library resource has been used to isolate long-sought functionally redundant auxin transporter genes (e.g. ABCB6, ABCB20; Zhang et al., 2018). Approaches to circumvent the inherent limitations of forward genetic screening with amiRNAs are developed in the present study. As a first step, it is recommended to rescreen the next generation to identify robust and reproducible phenotypes in individually isolated putative mutant lines. As a second step, the amiRNA sequence of confirmed mutant lines needs to be determined (see Methods). AmiRNA sequences linked to the observed phenotypes are retransformed and testing over 10 independent lines for the phenotype is recommended. Alternatively, amiRNAs which target a subset of the initially predicted targets can be used to narrow down the causative genes (e.g. Figure 3). For cases where only two to three genes are targeted T-DNA lines or CRISPR/Cas9 mutants can be used to narrow down the genes relevant for the phenotype.
Over 95% of the amiRNAs in this library were designed to target only two to five genes (Hauser et al., 2013), meaning that identification of causal genes is facilitated. Using the above approach, we report on two newly identified mutants: (i) amiRNA lines targeting three genes encoding avirulence induced gene 2 like proteins show an ABA insensitive seed germination phenotype. (ii) amiRNA lines targeting two proteasomal subunits show insensitivity to low CO2 induced stomatal opening. Further analyses of the two targeted genes in this amiRNA line, suggest that stronger T-DNA alleles result in lethality. This indicates the usefulness of the generated amiRNA lines for forward genetic isolation of higher order mutants that would be lethal upon knock out. Our data suggest that the wild-type expression levels of two alpha subunits of the 20S proteasome, α([0-9]+) and α([0-9]+), are required for fully functional stomatal opening mediated by physiological stimulation. This indicates that the proteasomal subunits are likely controlling an unknown general negative regulator of stomatal opening. The amiRNA seed resource generated here provides a new and potent tool to identify redundant genes and also lethality causing higher order mutants in many biological processes in Arabidopsis.
In conclusion, the amiRNA library resource is well-suited for screening of phenotypes that can be easily verified in subsequent generations. This population may be best suited for screens that permit high throughput or medium throughput screening for phenotypes with a large dynamic range. Many such screens have been performed in classical Arabidopsis mutant screens that were not designed to identify functionally redundant genes.
Supplementary data
Supplemental Table 1. List of relevant primers used in this study.
Supplemental Table 2. List of relevant plasmids used in this study.
Supplemental Table 3. List of new amiRNAs designed and cloned in this study.
Supplemental Table 4. AmiRNA sequences and predicted target genes found in candidate plants.
Figure S1. The induction of RAB18 gene expression by ABA is lower in the amiRNA-AIGs line.
Figure S2. The expression of AIGL genes in amiRNA-AIGs lines.
Figure S3. The p9l22 amiRNA line has normal stomatal indices and density.
Figure S4. The expression of PAB1, PAB2 and PAG1 genes in amiRNA lines.
Acknowledgements
Seeds for the over 14,000 T2 amiRNA lines described here are being made available to the Arabidopsis Biological Resource Center (ABRC) for screening by the research community (order numbers: CS99427, CS99428, CS99429, CS99430, CS99431, CS99432, CS99433, CS99434,CS99435, CS99436). We thank Dr. Jianyan Huang, Kellie Tao Kim, Wilma Lee, Sandra Vogel, Marianne Kreusch and Elly Poretsky for help in the transformation of the amiRNA library into Arabidopsis, and generation of the lines and support during the various stages of the screen for novel phenotypes. This research was funded by grants from the National Science Foundation (MCB1616236) and the National Institutes of Health (GM060396-ES010337) to J.I.S. and was in part supported by grants from the Israel Science Foundation (1832/14) and a European Research Council Starting Grant (757683 - RobustHormoneTrans) to E.S. P.H.O.C was supported by a Ciencias sem Fronteiras/CNPq fellowship. G.D. was supported by an EMBO long-term postdoctoral fellowship (ALTF334-2018).