CELLULAR MECHANISMS OF SELF-PROTECTION AGAINST DROUGHT-INFLICTED DAMAGES IN WHEAT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1012087
• Grant No.: 2017-67013-26200
• Cumulative Award Amt.: $410,000.00
• Proposal No.: 2016-10480
• Project Start Date: Feb 15, 2017
• Project End Date: Feb 14, 2021
• Grant Year: 2017
• Program Code: [A1152]- Physiology of Agricultural Plants

Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001

Performing Department
Institute Of Biological Chem

Non Technical Summary
Global climate change is predicted to reduce rainfall and curtail food production in the coming years. Hence, the ability to feed the growing human population critically depends on the availability of crop varieties with increasing drought tolerace. Drought induces the production of reactive oxygen species (ROS) in plants, which causes oxidative damage. We hypothesize that crop varieties that more efficiently manage ROS homeostasis may better protect cells from this damage and avoid subsequent yield losses. The complexity of ROS homeostasis, however, has impeded the identification of the contribution of individual genes that could be used in breeding programs. Furthermore, the measurement of ROS is difficult due to their chemical complexity and short life-time. Our group recently developed a technique that overcomes these challenges by quantifiying the peroxisome - an organelle and ROS sink - as a proxy for intracellular ROS. In this study, we will use wheat as a model system to: (1) measure the effect of drought on ROS homeostasis and crop yield in varieties that perform differently under water-limited conditions; and (2) determine the molecular mechanisms underlying the control of ROS homeostasis in drought-tolerant varieties. The results are expected to inform how the accumulation of ROS influences yield under drought, and what strategies cells use for maintaining ROS homeostasis. This knowledge will contribute to future security of food by improving the theoretical and technological bases for breeding drought-tolerant crops. The goals of this project align with Program Areas "Plant Health and Production and Plant Products" and "Physiology of Agricultural Plants."

Animal Health Component

40%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1549	1030	100%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1030 - Cellular biology;

Keywords

drought

ros stress

peroxisomes

spring wheat

tolerance mechanisms

Goals / Objectives
The long-term goal of our research program is to improve the drought-tolerance of wheat using a new technology for phenotyping the activity of ROS-scavenging machinery in plants. The goal of this project is to determine how efficiency of ROS scavenging influences yield and what strategies leaf cells use for maintaining ROS homeostasis under limited water availability. Our working hypothesis is that reduced production of ROS and more efficient mechanisms of ROS scavenging are essential components of drought avoidance and tolerance. We have developed a new approach for measuring peroxisomes using the small fluorescent probe N-BODIPY (Section 3) and demonstrated the potential of this approach for predicting yield. Here, we will take advantage of this discovery for phenotyping the activity of ROS-scavenging systems during drought stress in a set of wheat accessions with superior and/or inferior performance under drought. On the basis of the data shown in Section 3, we anticipate that a decreased abundance of peroxisomes in plant tissues reflects a lower concentration of ROS in cells, better fitness to drought and, consequently, higher yield. Wheat accessions with efficient ROS-scavenging machinery will be used in breeding programs for increasing drought-tolerance. Our goal will be achieved by pursuing two objectives.

Project Methods
We will analyze thirteen spring wheat varieties that have been selected based upon their average performance in areas throughout Washington State that receive low annual precipitation (Table 1). These data are publicly available from the Washington State University Wheat and Small Grains Variety Selection and Testing database (http://variety.wsu.edu/2013/index.htm).Table 1. List of selected spring wheat varieties.Variety Class* PI Number Characteristics Onas SWS PI 46796 Low proliferation of peroxisomesAlpowa SWS PI566596 High proliferation of peroxisomesHollis HRS PI 632857 High yield in droughtDrysdale Hard Breeding line High yield in droughtScarlet HRS PI 601814 High yield in droughtWA8190 HRS Breeding line High yield in droughtOtis HWS PI 634866 High yield in droughtPatwin HWS PI 643981 Low yield in droughtDayn (WA 8123) HWS Breeding line Low yield in droughtMcNeal HRS PI 574642 High yield in droughtAgawam HWS PI 648027 High yield in droughtLolo HWS PI 614840 Low yield in droughtIDO686 SWS Breeding line Low yield in drought *SWS, soft white spring; HRS, hard red spring; HWS, hard white spring.Wheat will be grown in 6.5 cm diameter, 25.5 cm height containers filled with Sunshine Mix LC1 potting soil (SunGro Horticulture, Bellevue, WA) and grown 16/8 hrs day/light cycle, 218 µmol PAR light and day/night temperature 22-23°C/16°C. Five plants (biological replicates) will be used per each control- and drought treatment. The order of pots with the same variety inside the growth chambers will be randomized to avoid a positional effect. Each pot will be irrigated with 250 ml of water (soil to water ratio 1:2) before planting the seeds. Then, pots will be irrigated daily with 125 ml of water. Starting from the tillering stage, water will be withheld from the experimental samples and plants will be transferred to the WSU Phenomics Center (https://labs.wsu.edu/phenomics/). Plant responses to drought will be monitored every 6 hrs by measuring chlorophyll fluorescence (Figure 12) and calculating photosystem II operation efficiency and Fv/Fm. The soil volumetric water content and stomata conductance will be measured daily using a Decagon Leaf porometer and a Soil Moisture Meter. Leaf samples will be collected at the values of soil volumetric water content that correspond to 20%, 15%, 10%, 5% and 0%. Simultaneously, leaf samples will be collected from the watered controls. Samples will be stored at -80°C. To compare the effect of drought on yield, watering will be resumed after the soil moisture level remains at 0% for eight days, and yield (the total weight and number of grains per spike) will be evaluated in control- and drought-stressed plants at maturity. Three independent replicates will be conducted in three-week intervals.Accumulation of H2O2 in leaves will be quantified using method of Loreto and Velikova, 2001.Peroxisome abundance in the total extracts from leaves will be measured using peroxisome-specific probe N-BODIPY.Activity of the peroxisome proliferation machinery will be estimated by measuring the transcription using qRT-PCR of a key peroxisome biogenesis gene PEX11.Intracellular ROS production will be detected by staining leaf cells with dichloro-dihydrofluorescein-diacetate (DCFH2- DA) according to Mhamdi et al., 2010. The presence of superoxide anion will be detected using dihydro-ethidium (DHE). Hydrogen peroxide will be detected by incubating leaves in 3′-(p-hydroxyphenyl) fluorescein (HPF).Chlorophyll fluorescence will be measured using a LI6400-40 pulse-modulated fluorimeter, and quantum yield of photosystem II (fPSII) will be determined using a multi-phase flash protocol. The rate of linear electron transport through photosystem II (PSII) will be calculated using equation Jf = fPSII*Abs*I*0.48, where Abs is leaf absorption, I is the incident irradiance, and assuming a relative excitation distribution to PSII of 0.48. Furthermore, the rate of electron transport required to sustain the photosynthetic carbon reduction and photorespiratory cycles (Jg) will be calculated from gas exchange measurements as Jg=(Anet+Rd)(4 Cc +8G*)/(Cc-G*) where Anet is net CO2 assimilation rate, Rd is dark-type respiratory rate, Cc is the chloroplastic CO2 partial pressure and G* is the CO2 compensation point in absence of dark-type respiration. The relationship between Jf and Jg will be used to determine the extent of photosynthetic electron transport that is consumed by ROS production.The DpH in different wheat varieties will be determined by electrochromic shift experiments deduced from absorption changes at 520 nm. Differences in the activity of the xanthophyll cycle will be monitored by reverse-phase HPLC, and the total content of PsbS will be determined by Western blot. Finally, the capacity and kinetics of qE that will be monitored by chlorophyll fluorescence quenching. Differences in the composition and quantity of ROS-scavenging pigments, such as tocopherols, will be measured in isolated thylakoid membranes by HPLC using the well-established procedure. The efficiency of the PSII repair cycle will be studied by measuring the relative content of the D1 protein by Western blot. These results will be complemented by measurements of PSII sensitivity to photo-damage by chlorophyll fluorescence quenching analysis.CAT activity will be determined by measuring the decomposition of H2O2 at 240 nm (extinction coefficient 39.4 mM cm-1). Peroxidase activity will also be measured according to the method published by Vanacker et al. (2000). rbate. Glutathione (total and oxidized) content in plant tissues will be measured in a neutralized extract by 5,5'-dithiobis(2-nitrobenzoic acid) according to Anderson (1985). Total ascorbate will be determined by spectrophotometry at 265 nm according to Rao and Ormrod, 1995.

Progress 02/15/17 to 02/14/21

Outputs
Target Audience:Target audience Over the four years our project encompassed educational, research, and extension activities that target the following audiences: 1. Plant research community. We demonstrated that plant response to drought could be evaluated by measuring abundance of peroxisomes in leaves. Peroxisome abundance informs is a cellular parameter. The role of cellular parameters in stress adaptation has been underappreciated thus far. Our findings enable further steps in studying mechanisms of drought adaptation on the cellular level. This direction of research will advance our knowledge about physiology of stress. 2. Wheat breeders. We developed a high-throughput assay for measuring peroxisome abundance and demonstrated that peroxisome abundance correlates negatively with yield. This knowledge is currently applied to identify genetic markers of peroxisome abundance in a project funded by Washington Grain Commission. The goal of this research is introducing markers of peroxisome abundance into breeding programs. 3. Post-doctoral scientist. This project provided training in plant stress physiology for post-doctoral scientists Marwa Sanad, Taras Nazarov, Glenn Turner and Tetyana Smertenko. Marwa Sanad now holds an independent position at the National Research Center in Egypt. Experience gained from this project promoted Marwa's career. 4. Graduate students. Graduate student Kathleen Hickey was trained in plant stress physiology and molecular biology. The topic of this project is the subject of Kathleen's dissertation. Kathleen is now in the third year of the graduate studies. 5. Undergraduates students. Two undergraduates were involved in the project, Austin Lenssen and Jessica Fisher. The project provided they with training in laboratory research and plant physiology. Austin would like to get involved in farming at Washington State. Jessica would like to continue working in the laboratory environment. 6. Stakeholders. We regularly informed members of the Washington Grain Commission and wheat growers at the Washington state about our research by participating in the annual review meetings and by publishing popular articles in the magazine Wheat Live. 7. Members of general public. We publicized our work through press releases and podcasts with the aims to increases awareness of drought stress and inform about solutions for ameliorating the impact of stress on yield. Changes/Problems:The progress during 2020 was hindered by COVID-19 pandemic. As the disease was spreading, the Washington State University as well as Washington State Government took measures to minimize the risk to the employees. All laboratory activities were extremely restricted from the middle of March until the beginning of May. During the remaining period, laboratory activities were restricted by the extreme social distancing measures. As a consequence, it was not possible to measure the composition and quantity of ROS-scavenging pigments, such as tocopherols. Another problem was associated with cancellation of conferences. Both University undergraduate research shows and the Annual Meeting of the American Society of Plant Biologists were cancelled or moved to virtual platforms, which complicated dissemination of the knowledge and getting feedback on our results. What opportunities for training and professional development has the project provided?1. Three undergraduate students, Jessica Fisher, Austin Ross, and Laura Wilson were trained in laboratory techniques, plant physiology, data analysis, and data presentation. Laura Wilson is the first-generation undergraduate from Walla-Walla community college. It was Laura's first experience in the lab. Students were involved in collecting leaf material from the drought-stress trials, measuring leaf stomatal conductance and hydrogen peroxide content in leaves, assaying activity of catalase, ascorbate peroxidase, super-oxide dismutase, and guaiacol peroxidase in leaves from control and drought-stressed spring wheat varieties. Jessica Fisher and Austin Lenssen presented a poster about her work at the Washington State University annual undergraduate research show and each won a prize for their work. Participation at the showcase provided Jessica and Austin with experience to defend their data in front of three judges. 2. Graduate student, Kathleen Hickey was trained in laboratory work, presenting results, data analysis, preparing figures for the manuscript and writing reports. Kathleen also prepared and presented a poster about this project at the 2019 annual meeting of the American Society of Plant Biologists in San Jose. This was Kathleen's first experience at the international meeting. During the meeting Kathleen not just informed her peers about the progress with the project, but also got a valuable feedback on her results. 3. This project also provided career opportunity for four post-doctoral scientists who worked par-time on the project Marwa Sanad, Tars Nazarov, Glenn Turner and Tetyana Smertenko. Marwa Sanad now holds an independent position at the National Research Center in Egypt. Experience gained from this project promoted Marwa's career. 4. PI Smertenko attended Plant and Animal Genome Congress in 2018 and Annual Meeting of the American Society of Plant Biologists in 2018. Both meeting provided an outstanding creed development opportunity for learning new technologies, getting feedback on the results, and networking with other scientists. How have the results been disseminated to communities of interest?The results of our work were disseminated as described below. 1. Publishing four papers in peer-reviewed journals, one manuscript is accepted, ands two manuscripts are in preparation. 2. Presenting posters at the annual meetings of the American Society of Plant Biologists in 2018 and 2019, and Plant and Animal Genome Congress in 2018. We were scheduled to present posters at the 2020 annual meetings of the American Society of Plant Biologists but the event was cancelled due to the COVID-19 pandemic. 3. Presenting posters at the Washington State University undergraduate research showcase in 2018 and 2019. There was a plan to present a poster at this show in 2020, but the event was cancelled due to the COVID-19 pandemic. 4. PI presented a talk at the 2018 Plant and Animal Genome Congress, seminars at the Noble Foundation (2018), Department of Crop and Soil Science of the Washington State University (2019), College of Horticulture & Forestry Sciences, Huazhong Agricultural University, Wuhan, China (2019), Pacific Northwest National Laboratory, Department of Energy, Richland, WA (2020). 5. PI presented results of his work at the annual research review of the Washington Grain Commission in 2017, 2019, and 2020. 6. Two popular articles were published in the magazine Wheat Life (2018 and 2020). 7. Three WheatBeat podcasts were recorded (2018, 2020, 2020) and published on the Washington State University website. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Phenotyping drought stress We compared drought response in 12 spring wheat varieties using high-throughput phenomics. Out of 12 varieties, Agawam, Drysdale, Hollis, Lolo, Patwin 515, and Onas exhibited distinct response to drought. Three varieties Hollis, Drysdale, and Patwin515 were selected for the next drought stress experiment. In addition to measuring the chlorophyll fluorescence parameters, flag leaf material was collected each day until the soil volumetric water content (VWC) decreased to 0%, which took 9 days. On day 9 plants exhibited typical signs of water stress: leaf wilting and yellowing. Five biological replicates were collected per each variety. The leaf material was stored at -80°C and used for biochemical assays. We measured impact of drought on development, yield structure and root growth in Hollis and Drysdale using 55 gallons U-line bins. Each bin was populated with five 2-week old seedlings. Five bins were setup per each variety: 2 well-watered controls and 3 for drought-stress. We analysed root architecture at week 3, 5, and 6. To assess the size of the root system, we measured two parameters: total root length and total root count. We observed no inhibition of root growth in Drysdale and almost complete inhibition of root growth in Hollis. Analysis of yield parameters revealed that Drysdale produced more tillers than Hollis, but each spike contained lower seed number. On the contrary, Hollis responded to drought by reducing the number of tillers while producing more seeds per spike. The flowering of Hollis was 7 days earlier than Drysdale. Measuring photosynthesis under drought stress Measurements were performed at 2% atmospheric oxygen to reduce the impact of photorespiration on quenching the excess of collected light energy at moderate (ca. 8% VWC) and severe (below 3% VWC) drought. Patwin515 exhibited the highest stomatal conductance values of all three. Under moderate drought, Drysdale was the least sensitive variety and kept stomatal conductance at the same level as the watered control. Photosynthesis was less sensitive to drought in Patwin515 than in Hollis and Drysdale. The ratio of net CO2 assimilation to stomatal conductance (A/gs) under both mild and severe drought was higher in Drysdale and Hollis, indicating more economic water usage as greater amounts of carbon were fixed per unit water lost. The ratio of energy created from linear electron transport through PSII (Jf) over the amount of energy required from electron transport to support gas exchange (Jg) was compared between genotypes and drought stress levels. A ratio larger than 1:1 indicates there is a great rate of electron transport than necessary to support the rates of CO2 assimilation and photorespiration. No differences were observed between varieties at each stress level indicating that under low photorespiratory conditions, energy generated from PSII was consumed similarly by all varieties. However, under severe drought and high light conditions Drysdale had greater non-photochemical quenching (NPQ) that appeared to be dissipating excess light energy prior the electron transport chain. Additionally, Patwin515 was able to maintain higher rates of photosynthetic carbon fixation than other cultivars under severe drought by maintaining higher stomatal conductance. Under ambient oxygen concentration (20.95%) analysis of the PSII photochemical quantum efficiency indicated higher electron flux in Hollis under control conditions followed by significant decline under severe drought. On the contrary, electron transport rate under normal conditions in Drysdale was lower, but then less affected by drought. This indicates that decline of electron flux in Hollis resulted from increased NPQ and was not due to downstream alternative electron sinks like carbon fixation and photorespiration. Higher capacity of NPQ could be the reason why proliferation of peroxisomes and activity of catalase is lower under drought in Drysdale. Hollis could be producing more ROS under drought due to lower NPQ. We also analyzed the electron flux through photosystem I. Lower non-photochemical loss of excited electrons due to reduced availability of acceptors in Hollis was accompanied by greater non-photochemical loss of excited electrons. This indicates that a greater proportion of harvested light energy in Hollis is used for ROS production relatively to Drysdale. Therefore Hollis would more likely to experiences greater oxidative damages. The photochemical energy use by photosystem I under drought was similar in both varieties. The sensitivity of PhiII from NPQ was similar in both varieties. It suggests that none of the varieties uses cyclic electron flux through photosystem I to reduce conversion of collected light energy into ROS. Measuring oxidative stress under drought Hydrogen peroxide content was used as measure of ROS accumulation in leaves of Hollis, Drysdale, and Patwin515. We found no significant differences indicating that under our experimental conditions plants prevented excessive accumulation of ROS. Next we explored mechanisms responsible for preventing the oxidative damages in Hollis and Drysdale. Peroxisomal abundance was higher in Hollis after the watering was withheld from 5 to 7 days. The Volumetric Water Content (VWC) decreased from 3.5% to 1 % during this period. Increase of peroxisome abundance was accompanied by higher activity of catalase on days 4 to 7. On the contrary, peroxisome abundance in Drysdale remained stable through the drought. This variety lacked a pronounced peak of catalase activity. Activity of superoxide dismutase (SOD) was higher at the earlier stages of drought in variety Hollis when VWC was between 8% and 3%. On the contrary, activity of SOD in Drysdale was higher during moderate drought stress (VWC between 8% and 1%). Activity of guaiacol peroxidase increased similarly in the course of the drought in both varieties. Activity of ascorbate peroxidase also increased in both varieties, however the peak in Drysdale was on day 5 and 6 whereas in Hollis the peak was on day 7. These results indicate robust ROS homeostasis in both varieties, but Hollis relies on peroxisomes to control ROS homeostasis whereas Drysdale uses cytoplasmic ROS scavenging system. Transcription of peroxisome biogenesis genes Peroxisome abundance in cells depends on the balance between peroxisome biogenesis and degradation. Peroxisomes biogenesis occurs through a fission process by proteins PEX11, FIS1A, DRP3A, DRP3B and DRP5B. As peroxisome abundance under drought differed between Hollis and Drysdale, we set out to analyse transcription of corresponding genes by qRT-PCR in leaves on the 5th day of drought and found that out of PEX11a, PEX11b, PEC11c, FIS1A, DRP3A, DRP3B and DRP57, only PEX11C was up-regulated by drought in both genotypes. Peroxisomes are known to contain ca. 300 different proteins (12-14). Plausibly, other peroxisome biogenesis genes could be transcriptionally up-regulated in response to drought, but we missed them in our assay. We analyzed transcription of all annotated peroxisomal genes in response to drought using 19 published RNA-Seq datasets from Arabidopsis thaliana, Oryza sativa, Sorghum bicolor, and Zea mays and found 75 to 120 genes encoding peroxisome proteins were differentially expressed. However, only PEX11 and catalase (CAT) were upregulated in all experiments. We also assessed activity of pexophagy pathway that is responsible for peroxisome degradation. As all types of autophagy, activity of pexophagy depends on the autophagic flux, which in turn is determined by transcription level of ATG8. We measured transcription of three ATG8 wheat orthologues and found only ATG8.4 was significantly up-regulated in Hollis in response to drought. It means autophagic flux is greater in Hollis.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Smertenko T., Turner G., Fahy D., Brew-Appiah R.A.T., Alfaro-Aco, R., Almeida Engler J., Sanguinet K. A., Smertenko A. (2020) Brachypodium distachyon MAP20 functions in metaxylem pit development and contributes to drought recovery. New Phytologist doi.org/10.1111/nph.16383.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Smertenko A., Clare S.J., Effertz K., Parish A., Ross A., and Schmidt S. (2020) A guide to plant TPX2-like and WAVE-DAMPENED2-like proteins. Journal of Experimental Botany, accepted.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Kathleen Hickey, Tom Sexton#, Magnus Woods#, Yunus Sahin, Jessica Fisher, Taras Nazarov, Karen Sanguinet, Helmut Kirchhoff*, Asaph Cousins*,Andrei Smertenko, (manuscript in preparation) Distinct mechanisms sustain yield under drought stress in spring wheat
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hinojosa L., Sanad M., Jarvis D., Steel P., Murphy K., Smertenko A.* (2019) Impact of heat and drought stress on peroxisome proliferation in quinoa. Plant Journal 99: 1144-1158. doi: 10.1111/tpj.14411.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sanad M., A. Smertenko A., and Garland-Campbell K.A. (2019) Differential Dynamic Changes of Reduced Trait Model for Analyzing the Plastic Response to Drought Phases: A Case Study in Spring Wheat. Frontiers in Plant Science 10:504. doi.org/10.3389/fpls.2019.00504.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Smertenko A.* (2017) Can Peroxisomes Inform Cellular Response to Drought? Trends in Plant Science 22, 1005-1007.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Thomas M Sexton, Balasaheb V Sonawane, Andrei Smertenko, Asaph B Cousins* (manuscript in preparation) Faster Soil Water Depletion Associated With Greater Water Use Efficiency in Drought Adapted Spring Wheat Cultivars
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Smertenko A., Sanguinet K. and J��ranta E. (2018) When navigating drought, peroxisomes may save the day. Wheat Life 61 (8), 57-59.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Smertenko A. and Reznik E. (2020) Dealing with oxidation: new path towards wheat plant health revealed. Wheat Life 63 (7), 44-45.

Progress 02/15/17 to 02/08/21

Outputs
Target Audience:Target audience Over the four years our project encompassed educational, research, and extension activities that target the following audiences: 1. Plant research community. We demonstrated that plant response to drought could be evaluated by measuring abundance of peroxisomes in leaves. Peroxisome abundance informs is a cellular parameter. The role of cellular parameters in stress adaptation has been underappreciated thus far. Our findings enable further steps in studying mechanisms of drought adaptation on the cellular level. This direction of research will advance our knowledge about physiology of stress. 2. Wheat breeders. We developed a high-throughput assay for measuring peroxisome abundance and demonstrated that peroxisome abundance correlates negatively with yield. This knowledge is currently applied to identify genetic markers of peroxisome abundance in a project funded by Washington Grain Commission. The goal of this research is introducing markers of peroxisome abundance into breeding programs. 3. Post-doctoral scientist. This project provided training in plant stress physiology for post-doctoral scientists Marwa Sanad, Taras Nazarov, Glenn Turner and Tetyana Smertenko. Marwa Sanad now holds an independent position at the National Research Center in Egypt. Experience gained from this project promoted Marwa's career. 4. Graduate students. Graduate student Kathleen Hickey was trained in plant stress physiology and molecular biology. The topic of this project is the subject of Kathleen's dissertation. Kathleen is now in the third year of the graduate studies. 5. Undergraduates students. Two undergraduates were involved in the project, Austin Lenssen and Jessica Fisher. The project provided they with training in laboratory research and plant physiology. Austin would like to get involved in farming at Washington State. Jessica would like to continue working in the laboratory environment. 6. Stakeholders. We regularly informed members of the Washington Grain Commission and wheat growers at the Washington state about our research by participating in the annual review meetings and by publishing popular articles in the magazine Wheat Live. 7. Members of general public. We publicized our work through press releases and podcasts with the aims to increases awareness of drought stress and inform about solutions for ameliorating the impact of stress on yield. Changes/Problems:The progress during 2020 was hindered by COVID-19 pandemic. As the disease was spreading the Washington State University as well as Washington State Government took measured to minimize the risk to the employees. All laboratory activiteis were extremely restricted from the Middle of March until the beginning of May. During the remaining period laboratory activities were restricted by the extreme social distancing measures. As a consequence it was not possible to measure the composition and quantity of ROS-scavenging pigments, such as tocopherols. Another problem was associated with cancellation of conferences. Both University undergraduate research show and the Annual Meeting of the American Society of Plant Biologists were cancelled or moved to virtual platform, which complicated dissemination of the knowledge and getting feedback on our results. What opportunities for training and professional development has the project provided?1. Three undergraduate students, Jessica Fisher, Austin Ross, and Laura Wilson were trained in laboratory techniques, plant physiology, data analysis, and data presentation. Laura Wilson is the first-generation undergraduate from Walla-Walla community college. It was Laura's first experience in the lab. Students were involved in collecting leaf material from the drought-stress trials, measuring leaf stomatal conductance and hydrogen peroxide content in leaves, assaying activity of catalase, ascorbate peroxidase, super-oxide dismutase, and guaiacol peroxidase in leaves from control and drought-stressed spring wheat varieties. Jessica Fisher and Austin Lenssen presented a poster about her work at the Washington State University annual undergraduate research show and each won a prize for their work. Participation at the showcase provided Jessica and Austin with experience to defend their data in front of three judges. 2. Graduate student, Kathleen Hickey was trained in laboratory work, presenting results, data analysis, preparing figures for the manuscript and writing reports. Kathleen also prepared and presented a poster about this project at the 2019 annual meeting of the American Society of Plant Biologists in San Jose. This was Kathleen's first experience at the international meeting. During the meeting Kathleen not just informed her peers about the progress with the project, but also got a valuable feedback on her results. 3. This project also provided career opportunity for four post-doctoral scientists who worked par-time on the project Marwa Sanad, Tars Nazarov, Glenn Turner and Tetyana Smertenko. Marwa Sanad now holds an independent position at the National Research Center in Egypt. Experience gained from this project promoted Marwa's career. 4. PI Smertenko attended Plant and Animal Genome Congress in 2018 and Annual Meeting of the American Society of Plant Biologists in 2018. Both meeting provided an outstanding creed development opportunity for learning new technologies, getting feedback on the results, and networking with other scientists. How have the results been disseminated to communities of interest?The results of our work were disseminated as described below. 1. Publishing four papers in peer-reviewed journals, one manuscript is accepted, ands two manuscripts are in preparation. 2. Presenting posters at the annual meetings of the American Society of Plant Biologists in 2018 and 2019, and Plant and Animal Genome Congress in 2018. We were scheduled to present posters at the 2020 annual meetings of the American Society of Plant Biologists but the event was cancelled due to the COVID-19 pandemic. 3. Presenting posters at the Washington State University undergraduate research showcase in 2018 and 2019. There was a plan to present a poster at this show in 2020, but the event was cancelled due to the COVID-19 pandemic. 4. PI presented a talk at the 2018 Plant and Animal Genome Congress, seminars at the Noble Foundation (2018), Department of Crop and Soil Science of the Washington State University (2019), College of Horticulture & Forestry Sciences, Huazhong Agricultural University, Wuhan, China (2019), Pacific Northwest National Laboratory, Department of Energy, Richland, WA (2020). 5. PI presented results of his work at the annual research review of the Washington Grain Commission in 2017, 2019, and 2020. 6. Two popular articles were published in the magazine Wheat Life (2018 and 2020). 7. Three WheatBeat podcasts were recorded (2018, 2020, 2020) and published on the Washington State University website. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Phenotyping drought stress We compared drought response in 12 spring wheat varieties using high-throughput phenomics. Out of 12 varieties, Agawam, Drysdale, Hollis, Lolo, Patwin 515, and Onas exhibited distinct response to drought. Three varieties Hollis, Drysdale, and Patwin515 were selected for the next drought stress experiment. In addition to measuring the chlorophyll fluorescence parameters, flag leaf material was collected each day until the soil volumetric water content (VWC) decreased to 0%, which took 9 days. On day 9 plants exhibited typical signs of water stress: leaf wilting and yellowing. Five biological replicates were collected per each variety. The leaf material was stored at -80°C and used for biochemical assays. We measured impact of drought on development, yield structure and root growth in Hollis and Drysdale using 55 gallons U-line bins. Each bin was populated with five 2-week old seedlings. Five bins were setup per each variety: 2 well-watered controls and 3 for drought-stress. We analysed root architecture at week 3, 5, and 6. To assess the size of the root system, we measured two parameters: total root length and total root count. We observed no inhibition of root growth in Drysdale and almost complete inhibition of root growth in Hollis. Analysis of yield parameters revealed that Drysdale produced more tillers than Hollis, but each spike contained lower seed number. On the contrary, Hollis responded to drought by reducing the number of tillers while producing more seeds per spike. The flowering of Hollis was 7 days earlier than Drysdale. Measuring photosynthesis under drought stress Measurements were performed at 2% atmospheric oxygen to reduce the impact of photorespiration on quenching the excess of collected light energy at moderate (ca. 8% VWC) and severe (below 3% VWC) drought. Patwin515 exhibited the highest stomatal conductance values of all three. Under moderate drought, Drysdale was the least sensitive variety and kept stomatal conductance at the same level as the watered control. Photosynthesis was less sensitive to drought in Patwin515 than in Hollis and Drysdale. The ratio of net CO2 assimilation to stomatal conductance (A/gs) under both mild and severe drought was higher in Drysdale and Hollis, indicating more economic water usage as greater amounts of carbon were fixed per unit water lost. The ratio of energy created from linear electron transport through PSII (Jf) over the amount of energy required from electron transport to support gas exchange (Jg) was compared between genotypes and drought stress levels. A ratio larger than 1:1 indicates there is a great rate of electron transport than necessary to support the rates of CO2 assimilation and photorespiration. No differences were observed between varieties at each stress level indicating that under low photorespiratory conditions, energy generated from PSII was consumed similarly by all varieties. However, under severe drought and high light conditions Drysdale had greater non-photochemical quenching (NPQ) that appeared to be dissipating excess light energy prior the electron transport chain. Additionally, Patwin515 was able to maintain higher rates of photosynthetic carbon fixation than other cultivars under severe drought by maintaining higher stomatal conductance. Under ambient oxygen concentration (20.95%) analysis of the PSII photochemical quantum efficiency indicated higher electron flux in Hollis under control conditions followed by significant decline under severe drought. On the contrary, electron transport rate under normal conditions in Drysdale was lower, but then less affected by drought. This indicates that decline of electron flux in Hollis resulted from increased NPQ and was not due to downstream alternative electron sinks like carbon fixation and photorespiration. Higher capacity of NPQ could be the reason why proliferation of peroxisomes and activity of catalase is lower under drought in Drysdale. Hollis could be producing more ROS under drought due to lower NPQ. We also analyzed the electron flux through photosystem I. Lower non-photochemical loss of excited electrons due to reduced availability of acceptors in Hollis was accompanied by greater non-photochemical loss of excited electrons. This indicates that a greater proportion of harvested light energy in Hollis is used for ROS production relatively to Drysdale. Therefore Hollis would more likely to experiences greater oxidative damages. The photochemical energy use by photosystem I under drought was similar in both varieties. The sensitivity of PhiII from NPQ was similar in both varieties. It suggests that none of the varieties uses cyclic electron flux through photosystem I to reduce conversion of collected light energy into ROS. Measuring oxidative stress under drought Hydrogen peroxide content was used as measure of ROS accumulation in leaves of Hollis, Drysdale, and Patwin515. We found no significant differences indicating that under our experimental conditions plants prevented excessive accumulation of ROS. Next we explored mechanisms responsible for preventing the oxidative damages in Hollis and Drysdale. Peroxisomal abundance was higher in Hollis after the watering was withheld from 5 to 7 days. The Volumetric Water Content (VWC) decreased from 3.5% to 1 % during this period. Increase of peroxisome abundance was accompanied by higher activity of catalase on days 4 to 7. On the contrary, peroxisome abundance in Drysdale remained stable through the drought. This variety lacked a pronounced peak of catalase activity. Activity of superoxide dismutase (SOD) was higher at the earlier stages of drought in variety Hollis when VWC was between 8% and 3%. On the contrary, activity of SOD in Drysdale was higher during moderate drought stress (VWC between 8% and 1%). Activity of guaiacol peroxidase increased similarly in the course of the drought in both varieties. Activity of ascorbate peroxidase also increased in both varieties, however the peak in Drysdale was on day 5 and 6 whereas in Hollis the peak was on day 7. These results indicate robust ROS homeostasis in both varieties, but Hollis relies on peroxisomes to control ROS homeostasis whereas Drysdale uses cytoplasmic ROS scavenging system. Transcription of peroxisome biogenesis genes Peroxisome abundance in cells depends on the balance between peroxisome biogenesis and degradation. Peroxisomes biogenesis occurs through a fission process by proteins PEX11, FIS1A, DRP3A, DRP3B and DRP5B. As peroxisome abundance under drought differed between Hollis and Drysdale, we set out to analyse transcription of corresponding genes by qRT-PCR in leaves on the 5th day of drought and found that out of PEX11a, PEX11b, PEC11c, FIS1A, DRP3A, DRP3B and DRP57, only PEX11C was up-regulated by drought in both genotypes. Peroxisomes are known to contain ca. 300 different proteins (12-14). Plausibly, other peroxisome biogenesis genes could be transcriptionally up-regulated in response to drought, but we missed them in our assay. We analyzed transcription of all annotated peroxisomal genes in response to drought using 19 published RNA-Seq datasets from Arabidopsis thaliana, Oryza sativa, Sorghum bicolor, and Zea mays and found 75 to 120 genes encoding peroxisome proteins were differentially expressed. However, only PEX11 and catalase (CAT) were upregulated in all experiments. We also assessed activity of pexophagy pathway that is responsible for peroxisome degradation. As all types of autophagy, activity of pexophagy depends on the autophagic flux, which in turn is determined by transcription level of ATG8. We measured transcription of three ATG8 wheat orthologues and found only ATG8.4 was significantly up-regulated in Hollis in response to drought. It means autophagic flux is greater in Hollis.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hinojosa L., Sanad M., Jarvis D., Steel P., Murphy K., Smertenko A.* (2019) Impact of heat and drought stress on peroxisome proliferation in quinoa. Plant Journal 99: 1144-1158. doi: 10.1111/tpj.14411.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sanad M., A. Smertenko A., and Garland-Campbell K.A. (2019) Differential Dynamic Changes of Reduced Trait Model for Analyzing the Plastic Response to Drought Phases: A Case Study in Spring Wheat. Frontiers in Plant Science 10:504. doi.org/10.3389/fpls.2019.00504.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Smertenko A.* (2017) Can Peroxisomes Inform Cellular Response to Drought? Trends in Plant Science 22, 1005-1007.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Thomas M Sexton, Balasaheb V Sonawane, Andrei Smertenko, Asaph B Cousins* (manuscript in preparation) Faster Soil Water Depletion Associated With Greater Water Use Efficiency in Drought Adapted Spring Wheat Cultivars
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Smertenko A., Sanguinet K. and J��ranta E. (2018) When navigating drought, peroxisomes may save the day. Wheat Life 61 (8), 57-59.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Smertenko A. and Reznik E. (2020) Dealing with oxidation: new path towards wheat plant health revealed. Wheat Life 63 (7), 44-45.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Smertenko T., Turner G., Fahy D., Brew-Appiah R.A.T., Alfaro-Aco, R., Almeida Engler J., Sanguinet K. A., Smertenko A. (2020) Brachypodium distachyon MAP20 functions in metaxylem pit development and contributes to drought recovery. New Phytologist doi.org/10.1111/nph.16383.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Smertenko A., Clare S.J., Effertz K., Parish A., Ross A., and Schmidt S. (2020) A guide to plant TPX2-like and WAVE-DAMPENED2-like proteins. Journal of Experimental Botany, accepted.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Kathleen Hickey, Tom Sexton#, Magnus Woods#, Yunus Sahin, Jessica Fisher, Taras Nazarov, Karen Sanguinet, Helmut Kirchhoff*, Asaph Cousins*,Andrei Smertenko, (manuscript in preparation) Distinct mechanisms sustain yield under drought stress in spring wheat

Progress 02/15/19 to 02/14/20

Outputs
Target Audience:During this reporting period we have reached to the brader interanational audience by published two papers in a peer-reviwed journals. We reached international scientific community by presenting results of our work at the Annual Meeting of the American Society of Plant Biologists. We information loaca growers by presentign our research at the Washingron Grain Commission Annual Review. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. During this period two undergraduate students, Jessica Fisher and Laure Wilson, were trained in laboratory techniques. Laura Wilson is the first-generation undergraduate from Walla-Walla community college. It was Laura's first experience in the lab. Both Laura and Jessica were involved in measuring activity of catalase, ascorbate peroxidase, super-oxide dismutase, and guaiacol peroxidase in leaves from control and drought-stressed spring wheat varieties. Jessica Fisher presented a poster about her work at the Washington State University annual undergraduate research show and won a prize for her presentation. Participation at the showcase taught Jessica in preparing the data, making the poster, and defending the poster in front of three judges. 2. One graduate student, Kathleen Hickey was trained in laboratory work, presenting results, data analysis, preparing figures for the manuscript and writing reports. Kathleen also presented poster about this project at the 2019 annual meeting of the American Society of Plant Biologists in San Jose. This was Kathleen's first experience at the international meeting. During the meeting Kathleen not just informed her peers about the progress with the project, but also got a valuable feedback. 3. This project also provided career opportunity for two post-doctoral scientists who both worked par-time on the project - Glenn Turner and Tetyana Smertenko. How have the results been disseminated to communities of interest?The results of our work were disseminated by: 1. Publishing two papers in a peer-reviewed journals. 2. Presenting posters at the professional meeting: annual meeting of the American Society of Plant Biologists. 3. Presenting a poster at the Washington State University undergraduate research showcase. 4. PI presented seminars at the Department of Crop and Soil Science of the Washington State University and at the College of Horticulture & Forestry Sciences, Huazhong Agricultural University, Wuhan, China. 5. PI presented results of his work at the annual research review of the Washington Grain Commission. What do you plan to do during the next reporting period to accomplish the goals?1. Finish comparing transcription of gene that control peroxisome proliferation under normal watering and drought stress in spring wheat varieties Hollis and Drysdale. Up to date was have designed primers, optimized PCR conditions, and prepared the required cDNA from the control and stressed samples. Measurements of the gene transcription will be accomplished during following several months. 2. Compare composition and quantity of ROS-scavenging pigments, such as tocopherols, in isolated thylakoid membranes from leaves of control and drought-stressed varieties Hollis and Drysdale by HPLC. 3. Compare efficiency of the PSII repair cycle in the above varieties under drought stress by measuring the relative content of the D1 protein in extracts from thylakoid membranes by Western blot. 4. Publish one research manuscript reporting outcomes of this project and one popular article about our work on the analysis of oxidative stress in plants.

Impacts
What was accomplished under these goals? 1. Analysis of ROS stress. During 2018 we started measuring impact of drought of the activity of ROS scavenging enzymes. In year 3 we completed these measurements with the focus on two varieties Hollis and Drysdale. These varieties were chosen for their different response to drought revealed in the experiments during 2017 and 2018. Drysdale responded to drought by reducing stomatal conductance, but producing more tillers and sustaining root growth. Reduction of yield under drought in Drysdale was caused by lower seed number per each spike. On the contrary, Hollis responded to drought by suppressing vegetative growth, both the number of tillers and root elongation. Stomatal conductance was also significantly reduced, but remained higher than in Drysdale. Peroxisome abundance in Hollis under drought stress was higher than that in Drysdale indicating that these plants experience greater oxidative stress. Hollis produced more seeds per spike under drought than Drysdale, but the number of spikes was reduced. In 2019 we conducted more detailed characterization of these varieties. The first experiment was measuring peroxisome abundance and activity of ROS scavenging system during successive stages of drought. Peroxisomal abundance was higher in Hollis after the watering was withheld from 5 to 7 days. Soil Volumetric Water Content (VWC) decreased from 3.5% to 1 % during this period. Increase of peroxisome abundance was accompanied by higher activity of catalase on days 4 to 7. On the contrary, peroxisome abundance in Drysdale remained stable through the drought. This variety lacked a pronounced peak of catalase activity. Activity of superoxide dismutase (SOD) was higher at the earlier stages of drought in variety Hollis when VWC was between 8% and 3%. On the contrary, activity of SOD in Drysdale was higher during the moderate stress (VWC between 8% and 1%). Activity of guaiacol peroxidase increased similarly in the course of the drought in both varieties. Activity of ascorbate peroxidase also increased in both varieties, however the peak in Drysdale was on day 5 and 6 whereas in Hollis the peak was on day 7. Concentration of hydrogen peroxide remained constant during the drought stress in both varieties. These results indicate robust ROS homeostasis in both varieties, but Hollis relies on peroxisomes to control ROS homeostasis whereas Drysdale uses cytoplasmic ROS scavenging system. Our results support hypothesis that peroxisome abundance can be used as a proxy for activity of peroxisomal ROS scavenging machinery. 2. Impact of drought on the photosynthesis. During 2018 we measured the impact of drought on the photosynthetic parameters at below-ambient oxygen concentration (2%). The outcomes were provided in the 2018 report. During 2019, we repeated these measurements in varieties Drysdale and Hollis under ambient oxygen concentration (20.95%). The electron transfer rate was higher in Drysdale under control conditions, but then was significantly inhibited under severe drought. On the contrary, electron transport rate under normal conditions in Hollis was lower, but then less affected by drought. This indicates that the electron flux decline in Drysdale resulted from increased non-photochemical quenching (NPQ) and was not due to downstream alternative electron sinks like carbon fixation and photorespiration. Higher capacity of NPQ could be the reason why proliferation of peroxisomes and activity of catalase is lower under drought in Drysdale. Additionally, Hollis could be producing more ROS under drought due to lower NPQ. We also analyzed the electron flux through photosystem I. Lower non-photochemical loss of excited electrons due to reduced availability of acceptors in Hollis was accompanied by greater amounts of oxidized donors. This indicates that a greater proportion of harvested light energy in Hollis is used for ROS production relatively to Drysdale. Therefore Hollis would more likely experience greater oxidative damage. The photochemical energy use by photosystem I under drought was similar in both varieties. The sensitivity of PhiII from NPQ was similar in both varieties. This suggests that none of the varieties uses cyclic electron flux through photosystem I as an adaptive mechanism to reduce conversion of collected light energy into ROS.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sanad M., A. Smertenko A., and Garland-Campbell K.A. (2019) Differential Dynamic Changes of Reduced Trait Model for Analyzing the Plastic Response to Drought Phases: A Case Study in Spring Wheat. Frontiers Plant Science 10:504. doi.org/10.3389/fpls.2019.00504.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hinojosa L., Sanad M., Jarvis D., Steel P., Murphy K., Smertenko A.* (2019) Impact of heat and drought stress on peroxisome proliferation in quinoa. Plant J. 99: 1144-1158. doi: 10.1111/tpj.14411.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: 1. Smertenko T., Turner G., Fahy D., Brew-Appiah R.A.T., Alfaro-Aco, R., Almeida Engler J., Sanguinet K. A., Smertenko A.* (2020) Brachypodium distachyon MAP20 functions in metaxylem pit development and contributes to drought recovery. New Phytologist. doi.org/10.1111/nph.16383.

Progress 02/15/18 to 02/14/19

Outputs
Target Audience:During the reporting period we have targeted the rollowing audiences: 1. Undergraduate students at the Washington State University. 2. International, national, and local graduate students and post-doctoral scientiests. 3. Framers and growers. 4. Agricultural policymakers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PI Andrei Smertenko attended annual meeting of the American and Canadian Societies of Plant Physiologists held in Montreal, July 14-18, 2018. This was an excellent opportunity to learn about new technologies in plant physiology, exchange ideas with peers, get feedback on our work and find out about progress with the research on crop stress biology. Undergraduate students Austin Lenssen and Jessica Fisher were trains in laboratory research methods: collection and handling leaf material, preparation of extracts from plant tissues, measuring protein concentration, ROS concentration, and activity of catalase, ascorbate peroxidase and guaiacol peroxidase. In addition, Austin learned how to measure stomatal conductance using leaf porometer. Austin also presented his work at the laboratory meeting. Both Jessica and Austin will present a poster at the undergraduate research fair in March 2019. How have the results been disseminated to communities of interest?The research was disseminated to the stakeholders and farmers at the Washington State through presenting our work at the Washington State University Dryland Farming Field Day, Lind, June 14, 2018. I collaborated with another faculty at the WSU, Dr. Karen Sanguinet and an artist Emilia Jååranta on a popular article "When navigating drought, peroxisomes may save the day" that was published in the August issue of Wheat Life (volume 6, pages 57-59). I recorded a podcast "Peroxisomes and Drought-tolerant Wheat", recorded in April 2018 and published on the Small grains website http://smallgrains.wsu.edu/wsu-wheat-beat-episode-18/. Our research was also disseminated to the members of the scientific community, students, and educators through the following activities. We presented a poster entitled "Impact of drought on photosynthetic activity, ROS homeostasis and yield in winter wheat varieties" at the American Society of Plant Biologists Annual Meeting, Montreal, July 14-18, 2018. We submitted a manuscript "Differential dynamic changes of reduced trait model for analysing the plastic response to drought phases: a case study in spring wheat" to the Frontiers in Plant Biology. The manuscript is currently under revision. What do you plan to do during the next reporting period to accomplish the goals?1. We will complete analysis activity of the ROS scavenging components in three spring wheat varieties grown under drought stress. 2. We will compare electron flow and content of anti-oxidant pigments in the spring wheat varieties exposed to drought stress. 3. We will compare CO2 assimilation and electron sinks in spring wheat varieties under drought-stressed and normal atmospheric oxygen.

Impacts
What was accomplished under these goals? 1. During the previous reporting period we have screened eleven spring wheat varieties for response to drought. These experiments were conducted in the phenomics chamber which allows automatic recording chlorophyll fluorescence parameters. Seedlings were subjected to drought starting at the tillering stage (Zadoks stage 21) by withholding the watering. Soil moisture was measured daily. NPQ (non-photochemical quenching), Fv /Fm (the quantum efficiency of open photosystem II centers); PiPSII (quantum yield of photosystem II photochemistry); qE (energy-dependent quenching); and qI (photoinhibitory quenching) were extracted daily from the images. According to changes in these varieties under drought, we choose three varieties: Hollis, Drysdale, and Patwin515. During the current reporting period we performed an experiment where these varieties were subjected to drought stress in the phenomics chamber as described above. In addition to chlorophyll parameters, we collected flag leaf material for biochemical assays each day of the drought stress until soil volumetric water content (VWC) decreased to 0%, which took 9 days. On day 9 plants exhibited typical signs of water stress: leaf wilting and yellowing. Five biological replicates were collected per each variety. We measured hydrogen peroxide content in leaves of Hollis, Drysdale, and Patwin515 as an indicator of oxidative stress at each time point. We found no significant differences. It means that under our experimental conditions plants prevented oxidative damages. Next we explored mechanisms that could be responsible for preventing the oxidative damages. First, we measured peroxisome abundance in Hollis, Drysdale, and Patwin515. The pattern was different: Hollis accumulated peroxisomes at the beginning of drought, whereas Patwin515 and Drysdale accumulated peroxisomes during later stages of drought when VWC was below 8%. We also started measuring activity of ROS scavenging enzymes. The first was guaiacol peroxidase (GPX) in Hollis. We found higher activity of GPX at the VWC below 3%. We will continue to analyze activity of the ROS scavenging enzymes in the third reporting period. 2. In a parallel experiment we collected a series of physiological parameters in plants measured at 2% oxygen. Under these conditions photorespiration is reduced, preventing excess energy from being quenched through this pathway. First, we compared stomatal conductance in these genotypes at moderate (ca. 8% VWC) and severe (below 3% VWC) drought. All three varieties were sensitive to the severe drought, but Patwin515 exhibited the highest conductance values of all three. Under moderate drought, Drysdale was the least sensitive and kept stomatal conductance at the same level as the watered control. Photosynthesis was less sensitive to drought in Patwin515 than in Hollis and Drysdale. The ratio of net CO2 assimilation to stomatal conductance (A/gs) under both mild and severe drought was higher in Drysdale and Hollis, indicating more economic water usage as greater amounts of carbon were fixed per unit water lost. The ratio of energy created from linear electron transport through PSII (Jf) over the amount of energy required from electron transport to support gas exchange (Jg) was compared between genotypes and drought stress levels. A ratio larger than 1:1 indicates there is a great rate of electron transport than is needed to support rates of CO2 assimilation and photorespiration. While this was observed under severe drought stress for all varieties, no differences were observed between genotypes at each stress level. This indicates that under low photorespiratory conditions, no differences were present in the way that reducing energy generated from PSII was consumed. However, under severe drought and high light conditions Drysdale had greater non-photochemical quenching (NPQ) that appeared to be dissipating excess light energy prior the electron transport chain. Additionally, Patwin515 was able to maintain higher rates of photosynthetic carbon fixation than other cultivars under severe drought by maintaining stomatal conductance. These diverse strategies employed by the genotypes measured here indicates that excess light energy is handled at multiple levels to minimize photodamage. We also analyzed activity of the ROS scavenging system: superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), GPX and peroxisome abundance. Each variety uses different strategies to control ROS homeostasis: APX in Patwin515; APX and GPX in Hollis; APX, CAT, and peroxisomes in Drysdale. In the next reporting period we will analyze photosynthesis and oxidative stress under atmospheric oxygen concentration to evaluate if photorespiration is an addition sink that can quench excess light energy. 3. During this period we also measured the impact of drought on the root growth in Hollis and Drysdale. Accessing deeper soil moisture by expanding the root system is a classical drought avoidance strategy. As Hollis and Drysdale are known to be drought-hardy and we did not observed profound differences in their photosynthetic performance under drought, we compared their root architecture under drought. Roots of wheat plants can reach several meters in depth. This factor hinders analysis of root architecture in the commonly used greenhouse pots. Analysis of roots in the field situation offers more comprehensive information. However, one growth season per year and variable weather conditions impose limitations on this approach. Here we set out to develop an approach that would allow comprehensive analysis of root architecture over several growth cycles during a single year under controllable growth conditions of a greenhouse. Instead of standard-sized pots, we used 55 gallons U-line bins. Each was filled with peat soil. The height of the soil bed was 80 cm. To image roots we inserted one transparent imaging tube in to each bin. In addition we also inserted two tubes in each bin for the soil moisture probe that would allow us to measure moisture at the middle and bottom of the bin. Bins were allowed to settle for one week and then watered for two weeks before planting the seedlings. In these experiments we planted five 2-week old seedlings of each variety per bin. Five bins were setup per each variety: 2 well-watered controls and 3 for drought-stress. Position of bins in the greenhouse was randomized. The bins were watered for 3 days and then watering of the drought-stress binds was stopped. We analysed root architecture at 3 different time points: week 3, 5, and 6. At the time points beyond 6 weeks, the contact between soil and root imaging tube was weak. Consequently root image quality was not sufficient for the analysis of root architecture. To assess the size of the root system, we measured two parameters: total root length and total root count. Both parameters correlated with each other at each week 3, 5, and 6 with R2=0.92, 0.86, and 0.94 respectively. The response of roots to the drought was different: no inhibition of root growth in Drysdale and almost complete inhibition of root growth in Hollis. We also collected leaf samples for analysis of peroxisome abundance at weeks 6 and 7. Peroxisome abundance was higher at both time points in Hollis, but was not affected in Drysdale. The yield per spike was higher in Hollis (1.4 g) relatively to Drysdale (1 g), however the number of spikes per plant was greater in Drysdale (17 vs. 11). As a consequence, the overall yield in these varieties was comparable. These findings mean that drought avoidance by increasing root system size and drought tolerance through ROS scavenging results in comparable yield.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Smertenko A., Sanguinet K. and J��ranta E. (2018) When navigating drought, peroxisomes may save the day. Wheat Life 61 (8), 57-59.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Marwa Naguib Mohamed Esmaail Sanad, Andrei Smertenko and Kimberly Garland-Campbell Differential dynamic changes of reduced trait model for analyzing the plastic response to drought phases: a case study in spring wheat
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kathleen Hickey, Austin Lenssen, Helmut Kirchhoff, Asaph Cousins, Karen Sanguinet, Andrei Smertenko. Capturing Drought-Avoidance Genotypes Using Peroxisome Proliferation Readout. Dryland Field Day in Lind, WA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Stasik O., Kovalenko M., Sokolovska-Sergiienko O., Kiriziy D., Hickey K., Nazarov T., Taran N., Batsmanova L., Sytnyk S., Oles V., Lakhneko O., Cooper K., Morgun B., Panchenko A., Smertenko A. Impact of drought on photosynthetic activity, ROS homeostasis and yield in winter wheat varieties. 2. American Society of Plant Biologists Annual Meeting, Montreal, July 14-18, 2018

Progress 02/15/17 to 02/14/18

Outputs
Target Audience:Scientific community Graduate and Undergraduate Students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral scientist Dr. Marwa Sanad, who was funded by the award attendend annual meeting of the American Society of Plant Biologists that was held in Hawaii, June 2017. During the meeting, Dr Sanad learned about new developments in research on drought, presented our results, and exchanged ideas with other scientists. It was an exciting professional deveopment opportunity for Dr. Sanad. PD Andrei Smertenko attended Plant and Animal Genome conference in 2018. During the meeting Dr. Smertenko learned about new technologies used for analysis of interactions between genotype and environment. The meeting provided an excellent opportunitey to seek feedback on the prograss with the project. How have the results been disseminated to communities of interest?The resultes were disseminated by presenting posters at two international meetings: 1. Sanad M., Hickey K., Fahy D., Garland Campbell K., and Smertenko A. "Dynamics of Peroxisome Proliferation during Plant Development and Abiotic Stress" Annual Meeting of the American Society of Plant Biologists, Hawaii, 2017. 2. Plant and Animal Genome annual meeting, San Diego, 2018. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will run a drought-stress experiment with selected four varieties. We will collect leaf material from each variety daily. At the same time we will measure photosynthetic parameters and stomata conductance. The tissue will be stored at -80C and used for biochemical and molecular analyses: 1. Measuring changes in reactive oxygen species (hydrogen peroxide) content in leave tissues. 2. Measuring peroxisome abundance. 3. Measuring transcription of peroxisome biogenesis genes. 4. Activity of ROS scavenging machinery. 5. Measure photosyntic paremeters and stomata conductance.

Impacts
What was accomplished under these goals? During the first reporting period of the project we analyzed photosynthetic parameters in 12 spring wheat varieties exposed to drought stress. The goal of this experiment was to determine whether drought would have different effect on the photosynthesis in these varieties. Seedlings were exposed to drought during tillering stage by withholding the watering. Control plants were watered daily. The soil volumetric water content was measured daily with Decagon soil moisture probe 5TE connected to the EM50 data logger. Chlorophyll fluorescence parameters were measured once per day using PSI (Photon System Instruments) high throughput phenotyping system. The drought response was assessed by three parameters: the quantum efficiency of open photosystem II centers (Fv /Fm); non-photochemical quenching (NPQ); photoinhibitory quenching (qI); and energy-dependent quenching (qE). Our experimental design took into account variability in the response of individual plants, unequal reduction of soil moisture in pots, and potential positional effect in the growth chamber. To account for potential difference between individual plants, we set 10 biological repeats (individual plants) per each variety for analysis under normal watering conditions or drought. Altogether, we measured 240 plants. As the phenotyping chamber fits 120 plants at a time, the lot was split in two sets of 120 plants. To account for potential uneven decrease of soil moisture level in individual pots, plants were grown in trays. Each tray contained 6 seedlings: 1 of each variety; 10 trays were watered normally and 10 trays were subjected to drought. Trays were placed randomly in the phenotyping chamber thus randomizing the positional effect. We found the most dramatic difference between the photosynthetic parameters of watered and stressed groups after the soil volumetric water content remained below 2.5% for 3 days. In both sets this time point corresponded to day 11 of drought. According to the values of the four parameters and available field yield data, we selected five varieties with distinct response to drought out of 12: Drysdale: high yield under drought, Fv /Fm slightly reduced; NPQ moderately reduced; qI significantly reduced; qE not affected. Hollis: high yield under drought, Fv /Fm significantly reduced; NPQ significantly reduced; qI significantly reduced; qE significantly reduced. Lolo: high yield under drought, Fv /Fm slightly reduced; NPQ significantly reduced; qI significantly reduced; qE not affected. Patwin 515: low yield under drought, Fv /Fm not affected; NPQ slightly reduced; qI significantly reduced; qE not affected. IDO686: low yield under drought, Fv /Fm not affected; NPQ not affected; qI slightly reduced; qE not affected. In the second year we conduct detailed analysis of biochemical, cellular, photosynthetic, and physiological parameters in these five varieties.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Smertenko A. Can Peroxisomes Inform Cellular Response to Drought? Trends Plant Science 22, 1005-1007.