Progress 06/01/20 to 05/31/24
Outputs
Target Audience: Throughout the academic year we regularly reached an audience of academic scientists and university students through regular seminars and lab meetings with members of the University of California at Berkeley and the USDA/ARS in Albany CA. We also reached a broader audience of students, scientists, biotech researchers, and plants breeders who regularly attendthe Annual Maize Genetics Conference where we gave seminars and presented posters from 2020 to 2024. In addition, we reached broader scientific audiences through invited in person seminars that PI Chuck presented at UC Berkeley in 2021, at Colorado State University in 2022, as well as virtual seminars presented to the Japan Botanical Society in 2021. More recently, we begun targeting a new audience of evolutionary biologists at these meetings due to our recent finding that KNOX/BLH complexes target known domestication loci. We targeted these various groups of people because they are the best prepared to translate our data and teach about gene function, selectgenes for improved agronomic traits, and integrateour genetic pathways with genomic datasets to effect positive breeding outcomes. For example, we connected with breeding specialists at the maize meeting who thought that our genes may be useful targets for the generation of elite germplasm with improved vascular characteristics and drought resistance. Changes/Problems:Objective 1. While we are confident that we have identified the overall changes in auxin physiology in the leaf veins of double mutants, we were not able to determine if vein fusion was altered in stems and how auxin influences it. Using fluorescent markers in year one, we were able to track overall leaf vein behavior, but had difficulty assaying complex vein fusions due to the multi-interconnected nature of stem vasculature. We tried to circumvent this problem by using clearing agents such as CLEARSEE to clarify the tissue and allow a 3D view of the entire stem architecture while retaining vein fluorescence. This protocol, however, failed on maize stems because they are very thick and do not allow penetration of the clearing agent. Thus, we could not track where the stem veins would fuse or not at nodal locations, especially in the mutants. In addition, correlating these events with the lack of, or presence of our auxin markers proved to be difficult as small changes in reporter expression were often seen independent of vascular events. Fortunately, a near 3D view of the stem vasculature could be reconstructed from serial paraffin sections on stem tissue, although this technique is not compatible with auxin reporter fluorescence. Also, we originally proposed to measure auxin levels in the double mutants, but abandoned this idea after we found from transcriptomics and fluorescent reporter analysis that only auxin response was altered in double mutants, not auxin transport nor metabolism. This proved to be fortuitous since no auxin metabolism, biosynthetic or transport genes we found to be true targets of BLH14 via ChIP-seq, so it is likely that we would have not seen any difference in auxin levels had we done this experiment. Objective 2. The original plan of isolating the BLH/KNOX protein complexes from shoots via mass spec proved to be more difficult than anticipated. A pilot mass spec experiment was performed for us in collaboration with Dr. Maria Jazmin Abraham Juarez at LANGBIO in Mexico on wildtype andBLH12/14mutants in years two to three. An initial co-IP pull down experiment using BLH antibodies did not detect any clear protein differences between one gram of mutant and wildtype shoot tissue when analyzed on a 2-D gel post proteolysis. We were advised that this result did not warrant performing a full mass spec sequencing experiment. It ispossible that by using more shoot tissue, a different tissue, or trying a better antibody could have alleviated this problem. However, our concurrent genomic efforts at analyzing the BLH/KNOX complex using ChIP-seq target genes containing binding sites for both proteins (Objective 4) proved to be more promising. This approach gave indirect proof that both proteins bind together as a complex on a genome wide scale, and thus this latter method became the focus of our efforts instead of doing more proteomics. Objective 3. We originally proposed to observe the effects of expression ofBLH12/14together with differentKNOXgenes such asKN1andLG3to determine if the different combinations display different phenotypes. As a test case, we produced transgenic lines expressing inducible versions ofBLH12andKN1in year one and planned to ultimately combine them together on the same transformation construct. In years two and three, however, anyattempts to combine theseBLHorKNOXtransgenes molecularly, or by crossing, were unsuccessful, likely due to lethality. Thus, in years three and four we had to pivot from making stable transgenic lines to performing transient induction of theBLH12/14transgenes in natural dominant KNOX over expression backgrounds. This is possible using an agro-injection assay in maize that we developed that depends on leaf type and injection location. The main issue with this protocol is that the transgenes tend to get silenced after one week, which is barely enough time to observe vein activity or differentiation. This injection technique did work in epidermal tissue overlaying vasculature, and we were able to observe nuclear transport of BLH12/14 only in naturalKN1maize over expressers. We were not able to determine if this co-expression led to re-differentiation of the epidermis to vascular identity, however, as the transgene was silenced several days later. Despite this, preliminary experiments indicate that auxin response is modified in these cells, consistent with results from our first objective. Objective 4. Due to technical issues, we had to perform ChIP-seq on floral tissue instead of shoots or leaves. During years one and two, Initial sequencing of BLH12/14 ChIP DNA from wildtype shoots generated multiple peaks of low confidence, most of which were present in the IgG negative control. Isolation of shoot nuclei usually resulted in very poor-quality chromatin heavily contaminated with starch granules. The pulldowns of both mutant and wildtype stem chromatin always gave similar nucleic acid amounts, instead of different amounts as expected, which is one of our prerequisites for moving ahead with library construction. Despite this, we sequenced the DNAs anyway, but found that they mainly consisted of low-quality random background reads. To address this problem, we chose to perform ChIP-seq on a different tissue. We focused on ear tissue which expresses both KNOX and BLH proteins at very high levels compared to shoots. We hypothesized that in shoots, most cells were terminally differentiated and do not express either BLH or KNOX proteins, making most of the available chromatin binding sites for either antibody inaccessible. We knew that in meristematic floral tissue a much higher proportion of cells expressing KNOX and BLH were present, with more chromatin binding sites available for antibody binding. This approach was a success, identifying over two thousand significant peaks that were missing in the negative controls, and uncovering 312 potential differentially expressed target genes. Finally, we had hoped to perform this same analysis with the BLH12 antibody, the expectation being that the same targets might be uncovered as BLH14 since both genes are functionally redundant. A ChIP experiment was performed with the BLH12 antibody and the resulting DNA was tested for enrichment for several target genes known to be bound by BLH14. Unfortunately, none of the targets we tested were significantly enriched, though this could be explained by the possibility that the targets differ between the two. More troubling, the DNA we recovered from the IP did not pass quality control standards set by the sequencing company due to low amounts and small size, so we chose to forgo sequencing the BLH12 ChIP DNAs. This might reflect the fact that the BLH12 antibody does not perform as well as the BLH14 antibody in vivo, something we already suspected from past experience using it on western blots. Unfortunately, we did not have enough time to generate and test a new BLH12 antibody before the end of the grant period. However, since our ChIP-seq ultimately used a full length BLH14 antibody that cross reacts with BLH12, it is highly likely that our successful BLH14 ChIP also pulled down many BLH12 targets as well. What opportunities for training and professional development has the project provided?This grant supported the partial salaries of one postdoctoral scientist, one research scientist, and one lab assistant. In addition, two summer interns were also trained to work on with the project. Through the entire project, lab assistant Emilio Corona has helped with maintaining the lab, performing plant care, and doing maize field work. In year one, the grant funded the salary of former post-doctoral scientist Dr. Zhaobin Dong who was able to attend the Maize Genetics Conference where he presented seminars and connected with other geneticists. This ultimately led to a job offer, and Dr. Dong left the lab in 2021 to start his own group at the CAU in Beijing. In year two, post-doctoral scientist Dr. Elena Shemyakina was recruited, and then master's student Lynne Hagelthorne in year three. Activities promoting the professional development ofDr. Shemyakina, and Lynne were accomplished during years two to four. Both scientists were new to maize genetics and learned how make use of maize as a model genetic system. In addition, Dr. Shemyakinagained bioinformatic skills and learned how to use Python programming to analyze ChIP-seq datasets to identifyBLH12/14target genes. Dr. Shemyakina also authored two posters that were presented at the 2022 and 2024 Maize Genetics Meetings in St. Louis and became a contributing member of the Maize Genetics community.One undergraduate student, Jenny Chau, was hosted in the lab in year one, and one high school student from Dublin High was hosted in year three. Jenny was trained to perform crosses in the corn field, isolate DNA, perform PCR, and perform confocal microscopy, all new skills.Her contribution to the project was so significant that she earned co-authorship on a forthcomingNatureGeneticspaper that will help with her graduate school applications. Finally, Lynne Hagelthorne was able to translate her new genetics experience to the biotech sector and obtained a job as a genomics specialist at a plant breeding company in 2023. How have the results been disseminated to communities of interest? Results have been disseminated to the Maize Genetics community and the general scientific community at large in the form of publications, seminars, and poster presentations. To date our project generatedfour scientific publications, four poster presentations, and one manuscript still in preparation. In addition, we gave five invited seminars about our project at U.C. Berkeley, Colorado State University, the Japan Botanical Society,and the Maize Genetics Meeting in St Louis. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. The goal of understanding the auxin physiology ofBLH12/14double mutants was achieved using a combination of immunolocalization, histology, and localization of auxin responsive fluorescent reporters. By assaying tagged auxin transporters such asPIN1AandSoPIN1in year one, or immunolocalizing them in aBLH12/14background in year two, we observed almost no difference between the mutant and wildtype, and both markers appeared to follow the vasculature and pool in the epidermis above the lateral veins as normal. However, through localizing fluorescent reporters for auxin response such asDR5:DsREDin years #1-3, we saw a clear difference around the bundle sheath cells that surround the lateral veins in double mutants where there was a reduction in fluorescence compared to wildtype. This is likely the site of initiation of intermediate veins that normally branch from lateral veins near leaf tips, structures that are reduced or missing in the double mutant. This conclusion was reached before the end of year #3, and was found to be consistent with our ChIP-seq and RNA-seq results that identify ARF transcription factor genes as targets of BLH12/14, but not auxin transport or metabolism genes. Because auxin response appears to be defective inBLH12/14, and not auxin flow or transport, the objective to measure auxin levels in the mutant compared to wildtype was deemed unnecessary since hormone levels overall were unlikely to differ. Objective 2. We confirmed that the KNOX homeodomain proteins LIGULELESS3 (LG3) and KNOTTED1 (KN1) are interactors with BLH12/14 in leaves versus stems respectively via pulldown and bi-molecular fluorescence complementation. Usingtobacco injected with split YFP tagged versions of BLH12/14 and KN1 and LG3 in years one and two, we showed that these complexes interact in vivo to facilitate nuclear transport, results that were confirmed by pulldown. This interaction supports the hypothesis that the interaction of the BLH12/14 complex with different proteins confers different functionalities. Given that LG3 is normally expressed in leaves, while KN1 is expressed in stems and meristems, it is likely that the BLH12/14 interactions with these two proteins in their respective tissues is the reason why BLH12/14 has two distinct functions in initiating intermediate veins only in leaves, while preventing their fusion only in stems. Objective 3. To determine the functional outcome of the different interactions of BLH12/14 with KN1 and LG3, we used both a transgenic approach employing Agrobacterium co-injection, but also a genetic approach using maize mutants. Although stable individualBLH12andKN1overexpression transformants were produced inSetariain year one, we could not co-express them together simultaneously after multiple attempts duringyears two and three, likely due to developmental toxicity from both transgenes interacting together. To bypass this problem, in years three and four we developed a new assay to test the functional outcome of BLH12/14 interactions with LG3 and KN1 using pre-existing dominantKN1andLG3maize mutants that naturally overexpress these genes in leaf vasculature. We co-injected BLH12 or BLH14 overexpression constructs in these dominant mutants, and only in mutant backgrounds couldBLH14::YFP travel to the nucleus, while in wildtype it could not. Thus, this approach ultimately confirmed that the BLH/KNOX interaction facilitates nuclear transport in vivo, but we were not able to determine if this interaction led to intermediate vein initiation or prevention of vein fusion since the epidermal cells were already terminally differentiated. Objective 4. Chromatin immunoprecipitation sequencing was completed using the BLH14 antibody, and hundreds of target genes were identified and are being validated. In years one and two, pilot ChIP experiments were attempted, but failed due to technical problems during nucleii isolation from shoot tissue. In years three and four, however, by switching to a new tissue and modifying the ChIP protocol, we were able to successfully perform ChIP-seq. From a pair of biological ChIP-seq replicates, 2,063 peaks overlapping peaks were found to be statistically significant. TheseChIP-seq results were filtered againstour earlier RNA seq dataset from youngBLH12/14shoots that had already been completed in years one and two, and 312 genes were found to be both bound and modulated. Of these genes, 208 overlapped with a list of KN1 bound genes previously identified by a prior ChIP-seq study. Thus, 208 targets were bound and modulated by both KNOTTED1 and BLH14, indicating that these genes may be targeted by BLH/KNOX complexes. The top two gene ontologies for these target genes were nucleic acid binding and transcription factor activity, indicating that the complex sits atop a regulatory hierarchy. In line with the conclusions of Objective #1 where only auxin response reporters were altered in BLH12/14 mutants, none of the KNOX/BLH targets we identified were involved with auxin transport or metabolism. However, six auxin response regulators were bound and modulated by both KN1 and BLH14, and four only by BLH14. These genes include several ARFs (auxin response factors) that function to activate transcription in the presence of auxin,and one AUX/IAA gene that negatively regulates auxin response. Unexpectedly, the maize domestication genegrassy tillers1 (gt1) was validated as a direct target, demonstrating thatBLHgenes may have played a role during maize domestication.gt1functions to repress axillary buds and is overexpressed in domesticated maize, but its function in patterning the stem vasculature was unknown.Moreover, a KN1 binding site in thegt1promoter identified from a previous ChIP study was found very close to the BLH14 binding site. This finding has opened a new avenue for understandingBLH12/14function, and we hypothesize that a BLH/KNOX complex plays an essential role in preventing the fusion of the stem vasculature to the newly initiating vasculature emanating from axillary buds. Hence, in modern domesticated maize where these buds are repressed, their lack of growth could be due to vein specific BLH12/14 activation of growth repressors suchgt1. Another BLH14/KN1 target gene that was uncovered wasZmYABBY14, a gene that in rice has recently been shown to be important for vascular differentiation at stem nodes. Interestingly, YABBY genes inArabidopsisand rice are known to be repressed by KNOX genes which contributes to leaf and node establishment in stems. This result demonstrates that BLH14 may prevent intermediate vein fusion in stem nodes by activatingZmYABBY14to promote internode identity and repress nodal identity. In sum, our accumulated data over the grant period provides a molecular and physiological framework for how BLH12/14 can affect intermediate vein initiation and prevention of vein fusion in stems. In leavesBLHgenes activateARFsto control auxin response which is known to be critical for vein initiation. This function may require the interaction of the KNOX protein LG3 which is widely expressed in most tissues. However, in the stem and meristem, BLH proteins interact with the KNOX protein KN1, forming a complex that binds to targets such as the domestication genegt1and the internode patterning genezmYABBY14. This former interaction may control fusion between vasculature within the stem to those from the axillary buds specifically at nodes, while the latter may specify nodal identity and axillary bud initiation. This mechanism provides an alternative means to prevent excess axillary bud growth, a condition that is necessary for ideal crop plant architecture.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Dong, Zhaobin, et al. "A regulatory network controlling developmental boundaries and meristem fates contributed to maize domestication." Nature Genetics (2024) in press
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Chuck, George et al. "tasselsheath4 is a novel domestication gene that controls developmental boundaries." 66th Annual Maize Genetics Conference (2024)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Shemyakina, Elena et al. "A KNOX/BLH homeodomain complex targets a major domestication locus." 66th Annual Maize Genetics Conference (2024)
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:We reached an audience of academic scientists, university students, private researchers at biotech companies, and breeders who attended theAnnual Maize Genetics Conference that took place in person at St. Louis MO on March 16-19, 2023. We also targeted unversity students and academic researchers at U.C. Berkeley through seminar presentations that took place on May 10, 2023. We targeted these groups of people because they are the best prepared to translate our data and effect practicaloutcomes in terms of spreading knowledge about gene function, and selecting genes for improved agronomic traits. For example, we connected with breeders at the meeting who thought that our genes may be useful targets for the generation of elite germplasm with improved vascular characteristics.We reached an audience of academic scientists, university students, private researchers at biotech companies, and breeders who attended theAnnual Maize Genetics Conference that took place in person at St. Louis MO on March 16-19, 2023. We also targeted unversity students and academic researchers at U.C. Berkeley through seminar presentations that took place on May 10, 2023. We targeted these groups of people because they are the best prepared to translate our data and effect practicaloutcomes in terms of spreading knowledge about gene function, and selecting genes for improved agronomic traits. For example, we connected with breeders at the meeting who thought that our genes may be useful targets for the generation of elite germplasm with improved vascular characteristics. Changes/Problems:Objective 1. While we are confident we have identified the changes in auxin physiology in the leaf veins of the double mutant, we still are having difficulty seeing similar changes around the stem vasculature. This is due to technical difficulties in predicting where the veins in stems will fuse, or understanding why they did not fuse at nodal locations. In addition, correlating these events with the lack of, or presence of our auxin markers proved to be difficult as small changes in reporter expression were often seen independent of vascular events. To circumvent this issue, we are employing clearing techniques of the entire stem using the CLEARSEE protocol that will preserve the fluorescence, but also allow us to see a 3D view of the entire stem vasculature. This will allow us to better correlate overall changes in reporter expression with vascular activity. Objective 2. Isolation of protein complexes using mass spec was performed by Dr. Maria Jazmin Abraham Juarez at LANGBIO on wildtype and BLH12/14 mutants. An initial co-IP experiment did not detect any clear protein differences between one gram of mutant and wildtype shoot tissue, and thus did not warrant performing mass spec. It ispossible that using more shoot tissue will alleviate this problem, or using floral tissue where it is far easier to isolate protein complexes, instead of shoot tissue. Objective 3. Due to the likely death of transformants expressing bothBLH12/14in combination withKN1orLG3, we had to pivot from making stable transgenic lines to inducing theBLH12/14transgenes in natural dominant KNOX over expressers in order to observe functional differences in vein activity. This is possible using a new agroinjection assay in maize that we developed that depends on leaf type and injection location. The main issue with this protocol is that the transgenes tend to get silenced after one week, which is barely enough time to observe intermediate vein initiation, or lack thereof. We hope to circumvent this issue by using dominant KNOX lines that already have vascular reporters in the mutant background such asDR5::DsREDwithwhich it will be much easier and faster to detect expression differences. Objective 4. Another major change we had to make is to perform ChIP-seq on whole shoot tissue instead of only stems or only leaves. Isolation of stem nuclei usually resulted in very poor-quality chromatin heavily contaminated with starch granules. The pulldowns of both mutant and wildtype stem chromatin always gave similar nucleic acid amounts, instead of different amounts as expected, and as required to move ahead with library construction. Moreover, ChIPon young leaves did not result in enough DNA to generate a library worthy of sequencing. Because it is far easier to perform ChIP-seq on young shoot tissue that includes both stem and leaf and identify targets from both, we decided to switch to using this tissue. While this will make it difficult to distinguish stem versus leaf targets as described in Objective four, we should be able to determine target gene tissue specificityby doing an RNA-seq experiment on BLH12/14 leaves versus stems. This is because it is far easier to isolate RNA from leaves and stems than it is to isolate chromatin.? What opportunities for training and professional development has the project provided?Activities promoting the professional development of post doc Elena Shemyakina were accomplished during the funding period. Dr. Shemyakinalearned how to use Python programming to analyze ChIP-seq datasets and identify BLH12/14 target genes. Dr. Shemyakina also authored a poster that was presented at the 2023 Maize Genetics Meeting in St. Louis and is slowly becoming a member of the Maize Genetics community. In addition, high school students from Dublin High School were hosted in the Chuck lab for summer training and mentoring during the funding period. The students helped perform crosses in the corn field, isolated DNA, and performed PCR. One student will be returning next summer for further training and plans to study biomedical research in college. How have the results been disseminated to communities of interest?Results have been disseminated to the Maize Genetics communityin the form of personal communications, and a poster presentationtitled "A Developmental Boundary Built By Mutual MicroRNA Repression." Results have been disseminated to the scientific community at large in the form of a manuscript titled "Novel Roles For A Maize Domestication Gene During Boundary Establishment" as well as through invited seminars at U.C. Berkeley. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Confirm that auxin response is the main defect behind the lack of intermediate vein initiation inBLH12/14leavesusing immunolocalization of several AUXIN RESPONSE FACTOR antibodies in a double mutant background. To visualize the presence or lack of vein fusion in stems using these same markers, we will employ CLEARSEE clearing methods that preserve fluorescence while allowing visualization of stem vasculature. Objective 2. Continue mass spec efforts for BLH12/14 in order to find new, or novel interactors that are not KNOX homeodomain proteins using a different tissue. Objective 3. Continue ourBLH12/14agroinjection functional assay on different dominant alleles ofKNOTTED1andLIGULELESS3that express KNOXgenes at higher levels, and thus are more likely to give rise to an altered vein morphology phenotype. We will also inject dominantKNOX alleles that already express auxin related vein markers such asDR5::DsREDbecause observing changes in reporter gene expression is often easier than observing a clear morphological change. Objective 4. Perform a new ChIP-seq experiment using the BLH12 antibody. Based on immunolocalization studies in both BLH mutant backgrounds, this antibody appears to cross react with BLH14, and thus may identify similar targets. Further affinity purification of the BLH12 antibody outside the conserved homeodomain will likely be necessary to make it more specific to fully accomplish this objective.
Impacts
What was accomplished under these goals?
Objective 1. The goal of understanding the auxin physiology of BLH12/14 double mutants has almost been achieved. By assaying auxin transporters such as PIN1A and SoPIN1 using fluorescent reporters and immunolocalization in a BLH12/14 background, we saw virtually no difference between the mutant and wildtype as both markers appeared to follow the vasculature and pool in the epidermis above the lateral veins. Using fluorescent reporters for auxin response such as DR5:DsRED, however, we saw a clear difference, specifically in the bundle sheath cells surrounding the lateral veins in the double mutant where there was a reduction in fluorescence compared to wildtype. This is likely the site of initiation of intermediate veins that normally branch from lateral veins near leaf tips, structures that are reduced or missing in the double mutant. This result is consistent with our chromatin immunoprecipitation results that identify AUX/IAA genes as targets of BLH12/14, but not auxin transporter genes. AUX/IAA genes function as repressors of the auxin response, and their activation by BLH12/14 in the bundle sheath does not allow these cells to respond to the auxin pools emanating from leaf bases near lateral veins, thus preventing intermediate vein initiation. We hypothesize that this situation is reversed, however, near leaf tips where BLH12/14 levels are low, allowing the veins to initiate. Because auxin response appears to be defective in BLH12/14, and not auxin flow or transport, the objective to measure auxin levels in the mutant compared to wildtype is unnecessary since the levels are unlikely to differ. Assaying the minute levels of auxin within leaf tips would have been technically difficult,required large amounts of tissue, and offered little clarity on BLH12/14 function. Objective 2. We confirmed the KNOX homeodomain proteins LIGULELESS3 and KNOTTED1 are interactors with BLH12/14 in leaves versus stems respectively via pulldown and co-immuno precipitation experiments. This interaction supports the hypothesis that the interaction of the BLH12/14 complex with different proteins confers different functionalities. Objective 3. We developed a new assay to test the functional outcome of BLH12/14 interactions with LIGULESS3 and KNOTTED1 using natural dominantKNOTTED1andLIGULELESS1maize mutants that overexpress the genes in leaf vasculature. This assay was necessary because lines overexpressing BLH12/14 together with either homeodomain could not be recovered asstable transgenic plants. This alternative assay involved injection of agrobacterium overexpressing eitherBLH12orBLH14marked with YFP into juvenile leaf tips fromeither dominantKNOXmutant in combination with a recessiveBLHmutant, and then following the fluorescent marker during intermediate vein initiation after three to seven days. Objective 4. Chromatin immunoprecipitation sequencing has been completed for BLH14 and target genes have been identified. 1,274 significant peaks have been identified thus far, 33 of which correlate with differentially expressed genes we previously found by RNA sequencing of BLH12/14 mutant shoots. As described in Objective one, these targets include AUX/IAA genes that affect auxin response and correlate with a repressive effect on intermediate vein initiation at leaf bases. Unexpectedly, the maize domestication genesteosinte branched1and grassy tillers1, were also found as targets that are downregulated in BLH12/14 mutants, indicating thatBLH12/14may have played a role during maize domestication. teosinte branched1and grassy tillers1 function to repress axillary branches and are overexpressed in domesticated maize. This finding has opened a new avenue for understanding BLH12/14 function, and we hypothesize that they played an essential role in preventing the fusion of the stem vasculature to the newly initiating vasculature emanating from axillary buds. In modern domesticated maize where these buds are repressed, their lack of growth could be due to vein specific BLH12/14 activation of growth repressors such as teosinte branched1.?
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Dong, Z. et al., "Novel Roles For A Maize Domestication Gene During Boundary Establishment"(2023)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Dong, Z., et al. "A Developmental Boundary Built By Mutual MicroRNA Repression"(2023) Maize Genetics Conference, St. Louis
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:During this period we reached an audience of maize genetics scientists, students, researchers, and breeders who attended the Annual Maize Genetics Conferencethat took place in person and virtually on March 31 to April 3, 2022 inSt. Louis Missouri. We also took part in a pre-conference Developmental Genetics workshop on March 31 at the same meeting. In addition, wereached students and faculty through ourseminar presentations tothe Plant Gene Expression Center in AlbanyCA, as well as the Department of Crop and Soil Sciences at Colorado State University at Fort Collins, CO. Changes/Problems:We did not anticipate how much tissue would be required for the mass spec and the ChIP-seq. While this was not a problem for the wildtype libraries, it was difficult to generate equivalent amounts of double mutant blh12/14 tissue because the stockis effectively sterile and can not be amplified, a fact which delayed all of our high throughput experiments. We have addressed this problem by amplifying segregating stocks of the single mutants that are more robust that the double mutant. We also spent considerable time having to optimizeour chromatin recovery from starchy differentiated stem and leaf tissue, and settled on using younger less differentiated tissue. Also, staffing shortages at QB3 at UC Berkeley have slowed down the ChIP sequencing and mass spec experiments. To alleviate the latter problem, we initiated a collaboration with an expert at sequencing immunoprecipitated protein complexes, Dr.Maria Jazmin Abraham Juarez at LANGEBIO. Finally, the transient overexpression of KNOX and BLH genes together is proving difficult to assay due to lack of observable phenotypes caused by non-functional proteins or transgene silencing. Producing better transformation constructsmay address this problem, but we are circumventing this by taking advantage of natural KNOX vascular specific overexpression mutants in maize to test the effects of BLH co-expression. What opportunities for training and professional development has the project provided?During the funding period one postdoctoral scholar and one undergraduate student werementored by PI Chuck. Dr. Elena Shemyakinais a recent hire and is new to maize genetics research. She replacespostdoc Dr. Zhaobin Dong who left the group last year to accept a professorship at CAU in Beijing. Elena participated in the annual Maize Genetics Conference in St. Louis Missourion March 31, 2022 and presented a poster. Undergraduate student Jenny Chauwas trained to do confocal microscopy for the project and recently graduated and started medical school. How have the results been disseminated to communities of interest?Scientific results have been disseminated to the community at large in the form of two peer-reviewed publications in 2022, one in theProceedings of the National Academy of Sciences, and the other in Science Advances.In addition, two posters describing our results were presented at the Maize Genetics Conference. Finally, PI Chuck presentedseminars describing the scientific results to the students and faculty ofthe Plant Gene Expression Centerand Colorado State University. Afterthe latter seminar, PI Chuck met with undergraduateand graduate students, faculty, and other educators todiscuss outreach andcareers in STEM fields. What do you plan to do during the next reporting period to accomplish the goals?Aim#1 - perform direct measurement of auxin inBLH12/14 leavesin areas where veins initiate. This requires amplification of mutant stocks since gram level quantities of leaf tip tissueare needed. Aim#2 - Perform mass spec of immunoprecipitated BLH12/14 complexes from different wildtype and mutant tissues, again using gram level tissue quantities since smaller quantities detected no difference. Aim#3 - Create a new 5' end KN1::GR fusion construct, produce transgenic plants and test induction in combination with BLH12/14 expression in other tissues. - In case the fusions are non-functional, we will perform transient co- expression experiments, via injection of BLH12/14 overexpression constructs on natural dominant KN1 and LG3 vascular overexpression mutantsin maize. Aim#4 - Sequence immunoprecipitated DNAs from shoot tissue and validate auxin response target genes. -Initiate ChIP-seq of chromatin isolated from leaf versus stem tissue and compare frequency of targets to whole shoot libraries.
Impacts
What was accomplished under these goals?
Impact statement There is an urgent need to breed crop plants with enhanced vascular characters in order to optimize their performance under stressful environmental conditions. Indeed, having high-density vasculature is critical for the function ofC4 photosynthesis, but the mechanism of how this is achieved in grasses is unknown. The goal of our project is to understand how acquisition of high vein density occurs in maize, and to use this knowledge to increase vascularization of C3 crop plants and improve their agronomic qualities. Two maize genes,BLH12andBLH14, are responsible for vein initiation in leaves while preventing vein fusion in stems, two distinct mechanisms that increase overall vein number. In the current reporting period we discovered how these genes affect hormone response, what other proteins they interact with, and what downstream genes they bind to. Taken together, these datagiveus a roadmapwith which to control vein number in any grass crop we choose. Accomplishments Objective 1.We completed an expression survey of several different auxin reporters in theblh12/14 double mutant background using immunolocalization and confocal microscopy. The auxin dual reporter line PIN1:YFP and DR5::DsRED was used for confocal analysis in the double, andantibodies made to the maize PIN1A, PIN1B and SISTER OF PIN1 (SoPIN1) auxin transport proteins wereused for immunolocalization. The results of this analysis confirmed that all vascular elements continued to either express the auxin reporters ortransport proteins in a normal pattern, and that their expression had little predictive value in establishing the sites of future intermediate vein initiation. The only difference we observed in the double mutant wasin the local response of cells to auxin (as marked by DR5::DsRED) in regions where intermediate veins should initiate. In the double, there wasa reduction in DR5 expression in the inner leaf tissues destined for vein initiation, indicating that BLH12/14 controls how cells respond to transported auxin emanating from the veins rather than controlling auxin transport itself. This is consistent with the fact that no auxin transporterswere found in the BLH12/14 RNA-seq datasets. These resultsindicatethat we should focus on target genes that are auxin responsive, or have auxin response elements in their promoters, and that such genes will be key to understanding how BLH12/14 functions. The ChIP-seq and RNA-seq data sets can now be filtered for such auxin response gene ontologies. Objective 2.We determined which KNOX proteins can interact with the BLH12/14 proteins. Thus far, we found thatall class one KNOX proteins that weassayed, including KNOTTED1 (KN1) and LIGULELESS3, are capable of interacting with BLH12/14 based on split YFP co-injection experiments in tobacco leaves. It has been previously established that this interaction occurs between the MEINOX domain of KNOX proteins and the BEL domain of BLH proteins, and we confirmed that these domains are essential for the interaction. Because these domains are similar in most class one KNOX and BLH proteins, we will likely see positive interactions forany KNOX/BLH co-injection combinations thatwe may try. Thus, we decided to cease these transient expression experiments and focused instead on isolating in vivo KNOX/BLH complexes using mass spec. Pilot immunoprecipitation experiments were started on wildtype shoot tissue using the BLH12 and BLH14 antibodies in collaboration with Maria Jazmin Abraham Juarez at LANGEBIO in Irapuato Mexico. Thus far, protein differences have not been detected between wildtype and mutant tissue from in-gel separated BLH14 protein complexes, although the KNOTTED1 protein was detected as predicted. Thisindicates thatthe immunoprecipitation protocol works fordetectingin vivo interactions, and that we simply need to increase the amount of tissueto be able to visualize them in-gel and sequence them. Objective 3.We were able to determine whether BLH and KNOX co-expression was able to alter vascular development in leaf tissue. We produced several confirmed, stable transgenicKNOX overexpression lines in Setaria, including an inducible UBI::KN1-GR to test in vivo interactions with several stable UBI::BLH12-GR linesthat we previously produced. The latter line produces mild yellowing of leaves upon dexmethasone induction of the transgene withno obvious vascular defects, and we hypothesized that such defects may only be observed when co-expressed with a binding partner such as KN1. Our previous analysis of BLH12/14::YFPtransient expression in tobacco and Setaria showed that the proteins remained cytoplasmic, and we hypothesized that co-injection with a class one KNOX gene may help them enter the nucleus where they may function. However, wetested induction of the KN1-GR transgene by germinating seeds on DEX media and still observed no leaf phenotype, which was surprising given the fact that KN1 overexpression produces a range of developmental defects in most plant species. Moreover, co-injection of KNOX/BLH overexpressing constructs in Setaria leaves did not confer obvious vascular defects, although the vascular cells werelikely terminally differentiated in this tissue.This may mean that the 3' end GR fusion renders the protein non-functional, and thus should be inserted in the 5' end, something we are constructing. Alternatively, this result may showthat the hypothesis is wrong, and that BLH12/14 together with KNOX proteins are not required for vascular patterning in leaves, although we have yet to show that this is true in stemand shoot tissue. Objective 4.We completed the expression profiling of the BLH12/14 double mutant and identified a suite of 298 significant differentially expressed genes that were down regulated, defining 48 different GO terms, one of which is response to auxin stimulus as predicted from the auxin reporter assay results. This data can now be used in conjunction with ChIP-seq data to filter and identify true target genes. We accomplished our goal of isolating a true in-vivo target gene that we can use to validate our libraries. We previously initiated ChIP-seq experiments on stem and leaf tissue after modifying our ChIP protocol to improve our chromatin recovery and reduce background. Previous attempts on older tissues were hampered by thedifficultly we encountered with separating chromatin from the highamount of starch plastids derived from differentiated cells, and by the fact that we did not have a true target gene to test for enrichment of our libraries. To address theseproblems, we used younger tissues that did not have many differentiated plastids and greatly improved on our ChIP-DNA recovery, while recovering little to no chromatin from the IgGand BLH12/14 mutant negative controls as expected. Using this new approach, we confirmed our first bona fide BLH12/14 target gene, the homeodomain gene GRASSY TILLERS1, that functions in axillary branch formation. This target makes biological sense in light of the lack of axillary branches in the BLH12/14 mutant, and we confirmed itsenrichment in the wildtype libraries compared to negative controls. In addition, we confirmed that the BLH ChIP QPCR peak is very close to the previously published KN1 ChIP peak in the GRASSY TILLERS1 promoter, which indicates that the BLH and KN1 proteins likely interact as a complex while binding to this target gene. This finding supports our main hypothesis for cooperativeBLH and KNOX function.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Klein, Harry, et al. "Recruitment of an ancient branching program to suppress carpel development in maize flowers." Proceedings of the National Academy of Sciences 119.2 (2022): e2115871119.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Xiao, Yuguo, et al. "Boundary domain genes were recruited to suppress bract growth and promote branching in maize." Science Advances 8.24 (2022): eabm6835.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Shemyakina, Elena, et al. "Distinctive features of maize vascular development." 64th Annual Maize Genetics Conference (2022)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Vajk, Angus, et al. "A disordered protein mediates sex determination and auricle development in maize." 64th Annual Maize Genetics Conference (2022)
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:During the reporting period we reached students and faculty at U.C. Berkeley and U.C. Davis, as well as scientists atthe USDA/WRRC in Albany CA via virtual seminars. In addition, our results were disseminated through seminars and poster presentations to students, scientists and maize breedersat the virtual annual Maize Genetics Conference. Changes/Problems:A major problem we did not anticipate was the difficulty in recovering stable inducible transgenic lines expressing BLH12/14 or KNOX genes. Although we were able to recover over a dozen inducibleBLH12:GR lines, we did not obtain any BLH14:GR lines, and only one KN1:GR line that does not appear to beinducible. It appears that inducible GR fusions to some of these genes may be lethal, which may necessitate movingthe GR portionfrom the 3' ends to the 5' ends of the BLH14 or KNOX constructs. If this does not solve the issue, we may have to rely on a different inducible system such as the heat shock promoter, or to usetransient expression protocols such as the agro-injection to assay if we want to observe the combined effects of BLH12/14plusKNOX expression on vasculature. A second major difficulty was optimizing the ChIP protocol for stem and leaf tissue, compared to the meristematic tissue we normally use. The main problem withthe former tissues is that they are almost fully differentiated and therefore contain a very large amount of starch containing plastids such as amyloplaststhat co-precipitate with, and contaminate the chromatin. This required several modifications and the use of more specialized ChIP protocols optimized for different species. Although we recently solved this problem for stem tissue, we may have to re-optimizeour protocols for differentiated leaveswhich will delay the identification of BLH12/14 targets in this tissue. Therefore, we anticipate that we will know what the stem targets of BLH12/14 are before we know the leaf targets. What opportunities for training and professional development has the project provided?Training activities During the funding period two students were trained and mentored by PI Chuck tocarryout the research activites described above, a U.C. Berkeley undergraduate named Jenny Chau, and a U.C. Berkeley Postdoctoral Scholar named Zhaobin Dong who was funded by the project. The bulk of the accomplishments were derived by Zhaobin Dong with assistance fromthe PI and Jenny Chau. Professional Development Jenny Chau graduated from the MCB department at UC Berkeley in May 2021 and is applying to medical schools. Dr. Zhaobin Dong addressed the Maize Genetics Community via virtual seminar described in the next section. He recently obtained a professorship at China Agricultural University in Beijing and left the Chuck lab in January 2021. How have the results been disseminated to communities of interest?Postdoc Zhaobin Dong gave a seminar at the 2021 Maize Genetics Meeting that was held virtually on March 8-12. The title of his talk was "The tasselsheath4 gene establishes developmental fields within floral phytomers via microRNA mediated mutual repression." PI George Chuck gave a campus wide special virtual seminaron Febuary 17, 2021 titled "Conserved and divergent agronomic genes ranging from grasses to trees." What do you plan to do during the next reporting period to accomplish the goals?Aim#1 -Perform immunolocalization on BLH12/14 mutants using other PIN antibodies to confirm that auxin transport is not affected. -Directly measure auxin levels using GC/MS of very young wild type andBLH12/14 leaf tips where intermediate veins initiate. Aim#2 -Perform in vivo pull down assays using BLH12/14 antibodies and testing for the presence of KNOX proteins. -Perform pilot mass spec assays of protein complexes pulled down with BLH12/14 to identify interacting KNOX, or other proteins. Aim#3 -Test the effects of transient expression of KNOX genes oninduced BLH12:GR transgenic lines with regards tovasculature -Create new inducibleGR:BLH14transgenic lines -Create new inducibleGR:KNOXlines, and cross them to BLH:GR linestoassay their combined effects on vasculature Aim#4 -Construct BLH14 ChIP-seq librariesfrom stem tissue and sequence -Perform pilot ChIP on stem tissue using a full length BLH12 antibody -Begin ChIP assays on leaf tissue with BLH12 and BLH14 antibodies using optimized protocols
Impacts
What was accomplished under these goals?
Impact statement There is an urgent need to breed crop plants with enhanced vascular characters in order to optimize their performance under increasingly stressful environmental conditions. Indeed, havinghigh densityvasculatureis critical for the function of C4 photosynthesis, a more efficient mode of carbon fixation compared to C3 photosynthesis. Despite this, the mechanism of how highdensity vasculature is achieved in C4 crop plants such as maize and sorghumis completely unknown.The goal of our project is to understand how acquisition of high vein density occurs in maize, and to use this knowledge to increase vascularization of C3 crop plants andimprove their agronomic qualities.Two maize genes that we work on,BLH12andBLH14, are responsible for vein initiation in leaves whilepreventing vein fusion in stems, two distinct mechanisms that increase overall vein number. In the last reporting periodwediscovered that they are able todo this by modulating the genetic response to hormones, and by directly interacting with other proteins. We aim to discover how this hormone response is controlled, and what these other proteins are.This knowledge, along with thecorresponding genes, gives us theblueprintwith which to control vein number in any grass crop we choose. Accomplishments 1. We found thatBLH12andBLH14 appear to promote local response to auxin as opposed to auxin transport We tested the hypothesis that auxin metabolism is altered by BLH12/14. Auxin transport and response was assayed in BLH12/14 double mutants by crossing them to the PIN1:YFP/DR5::DsRed dual auxin reporter system. PIN1:YFP expression reflects auxin transport while DR5::DsRed expression marks cells that respond to auxin. In a BLH12/14 background PIN1:YFP expression markedall veins in a manner similar to wild type, but major differences were seen with DR5::DsRed. In BLH12/14 leaveslarge gaps in DR5::DsRed expression were observed in leaf tips near where intermediate veins initiate. In wild type, DR5::DsRed expression marks the epidermal cells near the lateral veins, but inBLH12/14this expression is missing, leaving a large area of non-auxin responsive cells in the epidermis as well as several underlying cell layers.To confirm that auxin transport is not affected in the double mutant we performed immunolocalization. Preliminary results using a SISTER OF PIN1 (SoPIN1) antibody failed to mark this same gap of non-auxin responsive cells in the double mutant as lacking auxin transport. Other PIN1 antibodies, such as PIN1A and PIN1B, will also be used to confirm that auxin flow is not altered in the mutant. Thus, auxin flow per se does not appear to be the major cause forthe lack of intermediate veins in the double mutant. Rather,a lack of auxin responsiveness in groups of cells near the where those veinsinitiate appears to be the main cause. These results are supported by the RNA-seq gene ontologies described in section four, and inform ourfuture experimental plansmoving forward. 2. We found BLH12/14 to interact with KNOX proteins such as KNOTTED1 (KN1)and LIGULESS3 (Lg3) in order to facilitate their transport into the nucleus We initiated transient agro infiltration studiesto identify and characterize the protein interactors with BLH12 and BLH14. Based on the expression profiles of several class I KNOX geneswe chose two, LG3 and KN1,that were vascular expressed and whose tissue specificity overlap with BLH12/14 in leavesand stems respectively. We tried to examine the functional significance of their interactions by performing co-injection of combinations of fluorescently tagged proteins using transient assays in tobacco. We discovered that BLH proteins expressed alone rarely localized to the nucleus, but when co-injected with KNOX genes were able to be nuclear localized. This finding was confirmed using BLH plus KNOX split YFP constructs wherewe found that they were only nuclear localized when co-expressed. The work indicates that movement of the BLH12/14 proteins to the nucleus requires the nuclear localization signals from KNOX protein partners. This finding will be confirmed using mass spec on BLH12/14 protein complexes isolated from leaf and stem tissue and subsequent pull down experiments. 3. We produced stable BLH12:GR and KN1:GR transformants that result in non-complementary phenotypes. To test how BLH12/14 with their protein partners affect vasculature, we produced transgenic plants. Several stable BLH12:GR inducible transgenic lines was produced in Setaria and confirmed to bedexamethasone inducible via western blotting. After several weeks of growth on dexamethasone containing media until flowering, no phenotype was observed apart from a faint yellowing of leaves. An examination of cleared leaf vasculature found no major defects in patterning, including the intermediate veins. This finding was curious in light of the fact that BLH12 appears to have a profound effect on intermediate vein initiation in maize. In order to understand this result, we tested the ability of BLH12:YFP or BLH14:YFP fusions proteins to be transiently expressed in Setaria via agroinfiltration. Interestingly, we observed that the majority of the expression to be cytoplasmic instead of nuclear, similar to what we observed in Aim#2 in tobacco. This indicates that BLH12/14 requiresinteracting partners to enter the nucleus and perform theirfunctions, and these most likely are other KNOX proteins. Thus, overexpression of BLH12 will likely not produce any vascular effects unless they are co-expressed with otherKNOX genes. To test thiswe also produced KN1:GR stable transformants,but have not observed any protein induction or phenotypes to date. 4. We found differentially expressed genes in BLH12/14 double mutants affect sugar metabolism, transcription, and auxin response An RNA seq analysis of three biological replicates of BLH12/14 compared to wild type has been completed and analyzed. We discovered that the top three major gene ontologies (GO terms) that were uncovered (in order) were sugar metabolism and biosynthesis, regulation of transcription, and response to auxin stimulus. The sugar GO term is easily explained as a secondary effect of the reduction in intermediate veins in the double mutant, resulting in fewer bundle sheath cells where carbon is fixed and reduced into sugars. The second transcription GO term indicates that BLH12/14 most likely carry out their activities by targeting other transcription factors. Finally, the last GO term supports the finding in AIM#1 that BLH12/14 mainly affects auxin response as opposed to auxin metabolism. Thus, we expect that some of these differentially expressed auxin response genes willbe direct BLH12/14 targets once we perform chromatin immunoprecipitation(ChIP). These include several AUXIN RESPONSE FACTOR (ARF) genes and Small Auxin Up RNA (SAUR) genes. To find these direct target genes, we initiated a pilot ChIP experiment on chromatin isolated from wiltype stems compared to BLH12/14 stems using the BLH12 and BLH14 antibodies. A previous pilot experiment using a standard ChIP protocol yielded no difference in the amount of chromatin retrieved from mutant tissue compared to wild type. Based on past experience, we do not proceed with library construction if the chromatin enrichment is not higher in wild type compared to the mutant because the background peaks will be too high. To remedy this, we modified our ChIP procedure based on recently published protocols (Zhao et al., Plant Physiology 183, 2020) and was able to retrieve 2X more chromatin from wild type compared to the BLH12/14 mutant using a full length BLH14 antibody. This DNA is now ready for library construction and sequencing, and the peaks will be filtered against the RNA-seq results to identify direct targets. We will also begin to harvest leaf tissue for ChIP to compare leaf versus stem BLH12/14 targets.
Publications
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