Progress 05/01/24 to 04/30/25

Outputs
Target Audience:Unchanged with respect to previous progress report: The target audience of this project includes plant scientists and breeders. Targeted scientists include those interested in basic plant biochemical mechanisms (protease biochemistry and molecular biology), as well as barley breeders aiming to improve barley nitrogen use efficiency and grain protein concentration. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed a postdoctoral research associate, Dr. Igor Shchepetkin, to obtain training in biochemical methods including protein extraction from barley leaves, protein gel electrophoresis and blotting, protease activity assays using fluorogenic substrates, activity-based protein profiling, protein affinity purification using streptavidin-agarose, and mass spectrometry-based identification and quantification of purified proteins. How have the results been disseminated to communities of interest?Results from the first year of grant support were published in the Open-Access journal MDPI Plants, acknowledging NIFA funding: Schepetkin I.A. and Fischer A.M. (2024) Cathepsin B- and L-like protease activities are induced during developmental barley leaf senescence. Plants 13: 3009. https://doi.org/10.3390/plants13213009 Additionally, the postdoctoral research associate working on this project, Dr. Igor Shchepetkin, has participated in the 2025 Gordon Research Conference on plant proteolysis, presenting project data. What do you plan to do during the next reporting period to accomplish the goals?Plans for the next project period: Characterization of barley lines with single or multiple protease knockouts. These experiments will be based on PCR and sequencing to characterize gene modifications obtained, and use of biochemical protocols established thus far (activity assays, activity-based profiling) to establish the importance of targeted genes/proteins for proteolytic activities in senescing leaves. Relevant literature and transcriptomic data from the PI's lab indicate that, next to cysteine proteases, serine proteases including family S8 (subtilisins) and S10 (serine carboxypeptidases) may be functionally important for nitrogen remobilization from senescing leaves. We will therefore also apply activity-based profiling to that class of enzymes. For those experiments, we will utilize the serine hydrolase probe ActivX Desthiobiotin-FP (fluorophosphonate) containing a modified biotin tag. This probe specifically and covalently labels the active-site serine of enzymatically active serine hydrolases, including serine proteases. Analysis of serine proteases will profit of protocols established during the first two years of support for PLCPs. Candidate proteases will be cloned into E. coli expression vectors allowing production of fusing proteins with cleavable moieties. Fusion proteins will be affinity purified and fusion tags will be removed. Purified protease(s) will be enzymatically characterized at different pH range using fluorogenic substrates and specific inhibitors.

Impacts
What was accomplished under these goals? This project is focused on protein degradation in senescing barley leaves through protein-degrading (protease) activity for nitrogen export from leaves to developing sinks (barley seeds). During this reporting period the main results were obtained on the characterization of expression profiles of papain-like cysteine proteases (PLCPs) in barley leaves during the terminal phase of developmental (natural) leaf senescence. Plant material used in the study: Plants of barley variety "Gemcraft" were grown in a glasshouse of the Plant Growth Center at MSU. The plants were fertilized every alternate week until anthesis, starting at 2 weeks after germination. Leaves were harvested at week 6 after the onset of flowering (terminal stage of senescence) and stored at -20 oC. Results: The Z-FR-AMC and Z-RR-AMC cleaving activity of protein extract from 6-week barley leaves was eluted with a linear gradient of NaCl as a single peak with DEAE-Sepharose column chromatography. This cleaving activity was completely inhibited by the cysteine protease inhibitor E-64. Active fractions (based on the Z-FR-AMC/Z-RR-AMC enzymatic assay) were pooled and labeled with DCG-04, a biotinylated probe derived from the E-64 inhibitor that binds covalently and irreversibly to the active site PLCPs. . Labeled proteins were captured on streptavidin beads, washed, eluted with biotin, and separated by SDS-PAGE. Gels separated DCG-04-labeled PLCPs into two bands with molecular weights of 43 and 38 kDa. These bands were visualized by Coomassie staining, excised and digested with trypsin, followed by peptide extraction and analysis by LC-MS/MS. Based on quantitative MS analysis, eleven PLCPs were identified in the protein extract from 6-week barley leaves. These PLCPs belong to four subfamilies, including cathepsin L (HvPap-6 or RD21, HvPap-7, HvPap-8 or CP14 peptidase, HvPap-13, HvPap-14, and HvPap-17), cathepsin H (HvPap-12 or aleurain), cathepsin F (HvPap-1), and cathepsin B (HvPap-19 and HvPap-20). Among the identified PLCPs, HvPap-6 was the most abundant. Peptides corresponding to HvPap-6 were identified in both 43-kDa and 38-kDa spots in approximately the same quantity based on total spectrum count. Thus, our results indicate that active HvPap-6 is present in barley leaves in at least two active isoforms. With help from the Wisconsin Crop Innovation Center, a vector with four guide RNAs targeting four barley PLCP genes (HvPap-6, HvPap-8, HvPap-12, and HvPap-14) was constructed and used for barley variety 'Gemcraft' transformation, targeting four of the enzymes identified by mass spectrometry in late-senescing barley leaves (see 5, above). T1 seeds have been obtained, and will be analyzed for the presence of knockout mutations in the targeted enzymes.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Cathepsin B- and L-like protease activities are induced during developmental barley leaf senescence I. A. Schepetkin and A. M. Fischer Plants 2024 Vol. 13 Issue 21 Pages 3009 Accession Number: 39519927 PMCID: PMC11548477 DOI: 10.3390/plants13213009 https://doi.org/10.3390/plants13213009 https://www.ncbi.nlm.nih.gov/pubmed/39519927

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:The target audience of this project includes plant scientists and breeders. Targeted scientists include those interested in basic plant biochemical mechanisms (protease biochemistry and molecular biology), as well as barley breeders aiming to improve barley nitrogen use efficiency and grain protein concentration. Changes/Problems:Hiring a postdoctoral research associate has taken several months, with Dr. Igor Shchepetkin staring in late August, 2023. This has led to some delay in experiments planned for the first year of this project. What opportunities for training and professional development has the project provided?This project has allowed a postdoctoral research associate, Dr. Igor Shchepetkin, to obtain training in biochemical methods including protein extraction from leaves, protein gel electrophoresis and blotting, protease activity assays using fluorogenic substrates, and activity-based protein profiling. How have the results been disseminated to communities of interest?Dissemination will occur primarily through peer reviewed publications, and by sharing relevant data (ahead of publication) with Montana State University's barley breeding program. What do you plan to do during the next reporting period to accomplish the goals?Planned experiments will follow those laid out in the project proposal, including: 1. Active cysteine proteases will be isolated from barley leaves using DCG-04 and streptavidin-agarose. The affinity-purified proteases will be reduced, alkylated, and digested on-bead using filter-aided sample preparation with sequencing grade modified trypsin. Tryptic peptides will be analyzed by LC-MS/MS, and will be identified by database search. The purpose of these experiments is the identification of additional cysteine proteases (besides HvPaps 6, 8, 12, and 14) which may contribute to nitrogen remobilization from senescing leaves. 2. Candidate proteases will be cloned into E. coli expression vectors allowing production of fusing proteins with cleavable moieties. Fusion proteins will be affinity purified and fusion tags will be removed. Purified protease(s) will be enzymatically characterized at different pH range using fluorogenic substrates and specific inhibitors. 3. Characterization of barley lines with single or multiple protease knockouts. These experiments will be based on PCR and sequencing to characterize gene modifications obtained, and use of biochemical protocols established during year 1 (activity assay, activity-based profiling) to establish the importance of targeted genes/proteins for proteolytic activities in senescing leaves.

Impacts
What was accomplished under these goals? This project is focused on protein degradation in senescing barley leaves through protein-degrading ('protease') activity for nitrogen export from leaves to developing sinks (barley seeds). During this reporting period the main results were obtained on (1) total protein degradation and (2) characterization of protease activity in barley leaves during natural senescence. Plant material: Plants of barley variety "Gemcraft" were grown in a glasshouse of the Plant Growth Center at Montana State University. The plants were fertilized every alternate week until anthesis, starting at 2 weeks after germination. Flag and second leaves were harvested every week starting from the onset of flowering (week 0) until week 6 after anthesis, immediately shock-frozen in liquid nitrogen, and stored at -80oC. Each sample consisted of at least 10 flag or second leaves, and five independent samples were harvested. Leaves were ground to a fine powder in liquid nitrogen. Results: 1. We found that total protein content in leaf tissue decreased after week 3 until week 6 from the onset of flowering (n=4; data based on 4 samples for each time point). 2. Because ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant protein in leaves (~50% of soluble protein in barley), we also evaluated the degradation of large Rubisco subunit in naturally senescing barley leaves using antibodies specific for the N- and C-termini of the subunit and found that after week 3, a decrease of the Rubisco large subunit was evident in the protein fraction of the leaves investigated using both antibodies (data based on 3 independent immunoblots). 3. Using fluorogenic protease substrates (casein-FITC, Z-FR-AMC, and Z-RR-AMC) we found that proteolytic activity in barley leaves increased gradually from green (week 0) to senesced (week 5) leaves (n=4 for each time point). Additional application of protease inhibitors showed that specific inhibitors of cysteine proteases (E-64 and CA-074) potently inhibited the proteolytic activity with IC50 in the concentration range 11-65 nM for E-64 and 20-30 µM for CA-074, suggesting that mainly cysteine proteases control bulk degradation of proteins in barley leaves. 4. To further explore the activity of cysteine proteases in senescing barley leaves, an activity-based labeling approach was applied. After incubation of protein extracts from barley leaves with DCG-04, a biotinylated E-64 protease inhibitor, soluble proteins, prepared by fast acetone precipitation were separated by SDS-PAGE and the DCG-04-labeled proteases were detected using streptavidin-horseradish peroxidase. Two bands, with molecular weights of 43 and 38 kDa, were detected in the extracts with the 43 kDa band being most abundant (data based on 3 independent blots). These bands can be fully competed by pretreatment with E-64 and CA-074 prior to labeling, confirming that DCG-04 specifically labels cysteine proteases. 5. Moreover, using immunoblotting with anti-SAG12 antibodies we found that SAG12 cysteine protease was maximally expressed in barley leaves on weeks 4 until week 6 from onset of flowering (data based on 3 independent blots). 6. With help from the Wisconsin Crop Innovation Center, a vector with four guide RNAs targeting four barley cysteine protease genes (HvPap-6, HvPap-8, HvPap-12, and HvPap-14) was constructed and used for barley variety 'Gemcraft' transformation. Based on current knowledge, the enzymes encoded by these genes are likely to be functionally relevant for protein degradation in senescing leaves, and for nitrogen remobilization from senescing leaves to grains during the grain filling period. T1 lines for analysis will be obtained by late 2024, allowing their use during years 2 and 3 of this project. Thus, our experiments support that cysteine proteases, including the barley SAG12 homolog are major peptidases in barley leaves during natural senescence involved in nitrogen allocation from leaves to developing barley seeds, contributing to the control of grain protein concentration.

Publications