Clade A Type 2C Protein phosphatases are highly conserved negative regulators of the drought signaling pathway in plants . RNAi targeting of Clade A Type 2C Protein Phosphatase homologues in A. thaliana and B. napus started in our lab by Dianne Pater using the Brassica Database . The region targeted by the RNAi is a 400 bp highly conserved catalytic region common to both A. thaliana and B. napus Clade A Type 2C Protein Phosphatases . The RNAi construct for B. napus was designed under the inducible promoter prd29a, a strong abiotic stress inducible promoter which is especially responsive to drought due to the presence of multiple drought responsive elements . This drought inducible promoter was used to investigate the effects of knocking down PP2Cs under drought stress, while maintaining wild-type expected ABA response levels under non-drought conditions. In A. thaliana, RNAi PP2C expression was also driven by prd29a, to investigate whether the construct would confer a drought resistant phenotype in both a crop and a closely related model organism. Additionally, with the same RNAi sequence we generated a construct under the viral ubiquitous promoter p35S, which is predicted to allow for whole plant constitutive knockdown of Clade A PP2Cs. In A.thaliana 10 independent lines containing 35S-driven PP2C RNAi construct and 8 independent lines containing rd29a-driven PP2C RNAi constructs have been generated. Dianne Pater in our lab previously generated 12 independent lines containing rd29a-driven PP2C RNAi constructs for B.napus. Later, I generated 3 homozygous independent lines of both 35S and rd29a-driven PP2C RNAi constructs for A.thaliana by selecting for T3 non-segregating lines. To examine if knockdown of PP2Cs would affect plant water use efficiency in a drought assay, we established a low moisture drought assay.

In order to mitigate differences in soil from pot to pot, and establish a more uniform environment for studying water use efficiency between genotypes, we adjusted water weight . By monitoring the weight of pots and moisture levels of the pots,nft channel we circumvented issues of variability in water availability for our plants, along with establishing uniform stress conditions. We found that after a period of 6 weeks in non-drought conditions, there was no growth penalty in rd29a and 35S mediated PP2C knockdown mutants,indicating that our constructs posed no measurable effect in well watered conditions under the imposed conditions. After 3 weeks of water-limiting conditions, we found that our ABA hypersensitive knockout mutant hab1-1 abi1-2 pp2ca-1, and our rd29a and 35S mediated PP2C knockdown mutants showed no significant growth penalty as compared to their non-drought counterparts . However, wild type plants show a significant decrease in total biomass as compared to their non-drought equivalents . Our transgenic lines for both rd29a and 35S-driven PP2C knockdowns show enhanced drought tolerance without compromising growth in non-drought conditions. Effect of stress inducible knockdown of Type 2C Clade A Protein Phosphatases in ABA for B.napus ABA inhibition of seed germination was tested in rd29a-driven PP2C knockdown B.napus transgenic lines in 2 µM containing ABA plates . Out of the three lines I tested, it was observed that two of them had enhanced ABA hypersensitivity, while the third line was shown to have wild-type like ABA responsiveness in germination . One of our ABA hypersensitive line , showed a consistent delay of germination as assessed by cotyledon emergence in non-ABA conditions that was observed in three independent experiments . To examine if knockdown of PP2Cs would affect plant water use efficiency in a drought assay, we established a drought assay . In order to mitigate differences in water availability from pot to pot, and establish a more uniform environment for studying water use efficiency between genotypes we looked into using fritted clay, which has been demonstrated to dry down more evenly than soil.. We measured soil moisture levels to confirm effective drydown . In non-drought conditions, our B.napus rd29a mediated PP2C knockdown mutants show some variability in growth, with one line showing some growth penalty.

After 30 days of drought, we found that two of our lines may be more drought tolerant, however more replicates are needed to confirm the phenotype . Our transgenic lines show signs of being more drought tolerant, but more replicates need to be run to establish a clear phenotype.While the RNAi design for the knocking down of Clade A Type 2C Protein phosphatases was intended for B.napus, a crop plant in the same family as A.thaliana , the modelling was done through PP2C orthologues from B.napus and A.thaliana. Due to the similarities between A.thaliana and B.napus PP2Cs, we looked to test both species for drought related phenotypes at different growth stages. Here, we describe the drought and ABA responsiveness of our A.thaliana mutants. We found that both drought-inducible and constitutive PP2C knockdown lines yielded enhanced ABA responsiveness during germination, with constitutive knockdown lines showing increased germination stunting as compared to drought-inducible . Similarly, 35Smediated PP2C knockdown lines again showed higher ABA responsiveness through limitation of primary root growth in the presence of ABA as compared to the wild-type levels exhibited by the rd29a– mediated knockdown lines . The decreased ABA response in germination and root elongation of rd29a-mediated knockdown and 35S-mediated knockdown lines is consistent across each of the respective three linestested. Abiotic stress-inducible promoters such asrd29a or rd29b have cis-acting elements which allow induction of the gene under abiotic stress. Some of the more important cis-acting elements found in rd29a are ABA-responsive elements which are transcriptional activators responsive to ABA treatments, and Dehydration- responsive elements , which are responsive to drought and osmotic stress. It has previously been described that multiple ABRE confer ABA responsiveness inpromoters of ABA-inducible genes, however a single ABRE is insufficient to elicit an ABA response . The rd29a promoter is shown to have only a single ABRE, but ABRE interdependence with the Drought Responsive Element in rd29a has been shown to induce a weak ABA response . Further investigation using qPCR on these rd29a and 35S mediated knockdown lines to evaluate PP2C levels may be interesting to compare RNAi expression between the two promoters.

Notably, it would be interesting to look into AHG1 and AHG3 which are genes whose expression levels are higher in dry seeds .To determine if constitutive and stress inducible knockdown of PP2C would correlate with enhanced drought tolerance, we tested our mutants in a low moisture assay. While drought is an increasingly prevalent problem, areas of extreme drought remain only a small part of agricultural areas . As such, investigation of our mutants under constant low moisture stress would be more relevant to real world application than simply looking into survivability without water. We found that both drought-inducible and constitutive PP2C knockdown lines had no growth penalties in well-watered conditions . However, in low moisture conditions, both rd29a and 35S-mediated PP2C knockdown lines showed enhanced drought tolerance . This result is promising, because while our mutant lines both show enhanced drought tolerance, they do not show the growth penalty associated with PP2C knockout lines. Furthermore, by utilizing an assay more in line with the kind of drought more readily found in agriculture, results indicate that in A.thaliana , 35S and rd29a-mediated PP2C knockdown result in enhanced drought tolerance without any well-watered drawbacks.Interestingly,hydroponic nft contrary to ABA responsiveness in germination and root elongation we did not observe any noticeable differences between rd29a and 35S driven knockdown mutants. There is an increased response of the rd29a-mediated PP2C knockdown line in drought conditions as compared to ABA conditions. The enhanced drought response of rd29a mediated falls within our assumptions that multiple DRE would elicit a stronger drought response than a single ABRE would give as an ABA response. Interestingly, we did not find a significant growth penalty in well watered conditions for the ABA hypersensitive triple knockout mutant hab1-1 abi1-2 pp2ca . However, the triple knockout mutant did show enhanced drought tolerance . Further investigation of other homozygous rd29a- and 35S-mediated knockdown lines in a same assay would be required. Furthermore, using qPCR on these lines to determine PP2C expression levels and RNAi expression would be also be interesting, as we have no clear link between gene expression of PP2Cs and our phenotypes. Furthermore, comparing induction levels of rd29a under drought stress and ABA treatments would give us better insight into the different actions of DRE and ABRE in activating our knockdown. PP2C knockout lines abi1-1 and aba1-2 show lack of sensitivity to ABA, and increased stomatal density. It would be interesting to analyze stomatal density and index of our rd29a and 35S-mediated knockout lines to determine if there is any difference in stomatal development due to reduced expression of PP2Cs. Furthermore, PP2C knockout mutants have enhanced ABA-associated stomatal closure . Investigating transpiration rates of our drought-inducible and constitutive PP2C knockdown lines using the petiole feeding method described in Ceciliato et al,. would give us better insight into the responsiveness of our mutants to ABA. There is a clear link between osmotic stress in plants and activation of DRE , and while rd29a yields a mild response to ABA, rd29a has been shown to have a strong response to osmotic stress. Using whole plant gas exchange similar to the protocol described in Ceciliato et al,. 2019 with plants grown in hydroponics then subjected to an ABA or polyethylene glycol would give us better insight onto the differences in ABA and osmotic stress in rd29a-mediated PP2C knockdown.

While modelling the RNAi for B.napus PP2Cs was done through A.thaliana orthologues and incomplete sequence annotations for B.napus , differences between the species remain. Notably, in B.napus the sequence similarities between the RNAi and the targeted PP2Cs was found to be BnABI1 and BnABI2 , meaning we may have lowered targeting and expression of our RNAi in B.napus than in A.thaliana. As such, we looked into the different growth stages of B.napus to compare the rd29amediated PP2C knockdown in both B.napus and A.thaliana. We found that drought-inducible PP2C knockdown lines show variable responses to ABA, with two of our lines showing enhanced response to ABA , and one showing wildtype like ABA response . Interestingly, we found that one of our lines shows increased seed dormancy in control conditions . Seed dormancy is regulated by PP2Cs, with AHG3 and AHG1 being shown to heavily involved with DOG1, a positive regulator of seed dormancy . The DRE elements of the promoter for rd29a have been also shown to be strongly activated under cold stress . We speculate that this specific line which shows enhanced seed dormancy under control conditions may have been more affected by the vernalization process due to a stronger insertional event, leading to a stronger PP2C knockdown. By testing our seeds in qPCR for PP2C gene expression in both cold and ABA conditions, we may have a better insight into the responsiveness the rd29a promoter for in either condition. To determine if stress-inducible knockdown of PP2C would correlate to enhanced drought tolerance, we tested our B.napus mutants in a drought assay. We were uncertain of the degree of RNAi expression in B.napus transgenic lines. As such we sought to create an assay that would be most representative in differences in drought tolerance between genotypes. Previously in our lab, Dianne Pater had developed a protocol for rapid dry down drought assay using fritted clay. The advantages of this protocol was that the soil used was very uniform in its dry down , and created a stress that was well balanced between all genotypes in the pot. However, these conditions are not as effective in analyzing real-world adaptability of our mutants, as the protocol demands the soil be fertilized every other day, and mortality rate at the seedling stage is very high , resulting in low sample sizes. We found that through the fresh weight in our drought assay, that two of our lines showed a trend toward being more drought tolerant . However, one of these lines also showed a trend towards having some growth penalties under non-drought conditions . While the assay was effective in maintaining moisture levels at a stable level across post , high mortality rate of seedlings and stunting of growth as compared to soil conditions, render this assay’s results inconclusive.