Progress 06/01/20 to 05/31/24
Outputs
Target Audience:There have been two main target audiences: 1. Other scientists, both within the field of research and outside it, essentially photosynthesis researchers in the former case, and those interested in agricultural resilience in the second. 2. The private sector, both companies interested in maize improvement, and also companies interested in using the technology we developed in other crop species. Changes/Problems:As noted, Aim 1 was not completed because the collaborator left her positoin. Aim 3 because the genetic resource provided was not usable. Sometimes collaborations work out great, other times not so much. The good news is that this allowed us to fully focus on Aim 2, and more than complete it. Also because the Aims were completely independent, problems with Aims 1 and 3 did not impeded Aim 2. What opportunities for training and professional development has the project provided?There have been three levels of professional development provided: 1. The graduate student is pursuing thesis research on this project, which is coming with multiple opportunities to share the research and explain the linkage between foundational research and agricultural improvement. Through her various channels of mentoring, she is growing as a spokesperson and scientists, and is heavily involved in a range of science activities outside the laboratory. 2. The project has also benefited from part-time technical help. The Research Assistants who have contributed the project are recent college graduates who wish to gain additional experience prior to entering a graduate program or the workforce. The project is helping them to burnish their scientific credentials both at the lab bench and in the presentation space. 3. The project has hosted two undergraduate interns for 8-month stints, through a cooperative program with Univ. Waterloo. Upon returning to complete their undergraduate degrees, these students will be highly qualified for graduate or technical positions. In fact, the graduate student supported by this project was herself a Waterloo intern in this laboratory, prior to beginning her graduate studies. How have the results been disseminated to communities of interest?We have listed two publications. In collaboration withBTI's Translational Science Program, numerous conversations have occurred with potential industry partners regarding the technology and how to move further into the proof-of-concept stage. Publication of the patent is also an important outcome. The student, technician and intern have all presented public posters and in some cases (the gradudate student) attended national or international conferences to discuss their research. The PD and graduate student also speak to other labs - either the lab head or lab members - when technical questions arise or when collaborative possibilities arise. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim 1: Obtain further insight into improved performance in existing transgenic lines, focusing on the levels of photosynthetic metabolites. The metabolite analysis was performed and and some differences were observed between controls and transgenic lines of interest. The most interesting were consistent with known physiological phenotypes of the plants, e.g. an apparent reduction in the Rubsico side reaction. However, our collaborator also encountered difficulties with sample preparation and eventually left the institute, making it impossible to complete the project to statistical significance. Aim 2:Create and begin analysis of new transgenic maize designed to overexpress RCA or PPDK. This Aim has been the main thrust of our activity and has been completed more or less as planned. For Rubisco Activase (RCA), we proposed to express forms containing heat-tolerant Agave sequences both in vitro and in vivo. We did so and found that as expected, Agave RCA can activate maize Rubisco. We then generated the planned transgenic lines, and are in the middle of defining their growth and Rubisco activity phenotypes. So far, we can say with confidence that these foreign RCAs are not harmful to maize, and that we have early indications that under some heat stress conditions, they can improve performance. Regarding PPDK, we proposed to express a foreign protein (from Miscanthus) that could confer chilling tolerance, as Miscanthus is a chilling-tolerant C4 grass. We indeed created those transgenic lines, and in addition we obtained a maize ppdk mutant and used the Miscanthus transgene to complement the mutant. Thus, we have maize plants that rather than expressing ZmPPDK, express MgPPDK. This creates a very useful platform for PPDK manipulation. Concerning the phenotypes of maize plants expressing MgPPDK, we have not yet demonstrated chilling tolerance, probably because of the level of transgene expression, but we do have interesting indications that MgPPDK is more rapidly regulated than ZmPPDK in terms of their light-responsive phosphorylation/dephosphorylation cycle. We hope to exploit this trait by exploring how these complemented plants perform under fluctuating light where PPDK regulation is important, and which also more closely mimics outdoor conditions than the continuous daylight provided in greenhouses or growth chambers. Lastly, we have made the proposed cross between MgPPDK lines and high-Rubisco plants. These have shown a promising synergistic phenotype under chilling conditions, in preliminary experiments. Aim 3.Conduct field trials of transgenic lines to validate differences observed under more controlled conditions. We could not complete this Aim because the necessary genetic backgrounds could not be obtained. Field experiments with maize require a uniform genetic background, for which we initially require inbred transgenic lines. These were eventually delivered to us, but we found out that they did not express the transgenes, for reasons unknown. Therefore we were unable to produce the seed needed for field work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Eshenour, K., Hotto, A., Michel, E. J., Oh, Z. G., & Stern, D. B. (2024). Transgenic expression of Rubisco accumulation factor2 and Rubisco subunits increases photosynthesis and growth in maize. Journal of Experimental Botany, erae186.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Hotto, A. M., Salesse-Smith, C., Lin, M., Busch, F. A., Simpson, I., & Stern, D. B. (2021). Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones. Journal of Experimental Botany, 72(13), 4930-4937.
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:Target 1: Scientist interested in crop improvement, particularly as related to photosynthesis and carbon sequestration Target 2: Potential industry partners with capacity in maize improvement Target 3: Potential donors for offshoots of this research Target 4: Interested public audiences including those with an interest in agriculture and crop resilience Changes/Problems:Aim 1. Our main collaborator's lab is closing, making the specialized technology unavailable. Because the initial results were marginally significant, we elected to place our focus and limited funds in other areas, particularly Aim 2. Aim 2. No problems. Aim 3. We obtained the desired transgenic lines but with a delay of two years because the partners who were to create the original lines, were unable to do so. However, the Aim can now proceed as planned. What opportunities for training and professional development has the project provided?The postdoctoral fellow obtained her desired position in the biotech industry, validating her participation on this project as excellent preparation for such a position. The undergraduate participant received active mentoring and is completing her final year of undergraduate education at this time. Our hope is that she will remain in STEM and perhaps plant sciences. The graduate student is outstanding and is both receiving mentoring but also acting as a key collaborator to bridge different research groups with overlapping interests. By operating at such a high level she is gaining a broad skill set that will serve her well in the future. How have the results been disseminated to communities of interest?We have submitted our Raf2 results for publication and await a response from the editorial board. The graduate student has presented results at two meetings, one international, and met with several visiting scientists. Because of our new patent related to this research, the PD and graduate studenthavespent time with our Translational Science department seeking ways to advance the technology through partnerships, particularly to move it to trials in commercial germplasms. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Completed. Aim 2. The focus is on phenotyping and this is currently underway. We have the necessary materials and protocols/instrumentation in place. This should allow us to complete the major goals of the project in this Aim. Aim 3. The coming year will be spent phenotyping the LH244 transgenic lines and growing the top lines in winter nurseries to generate F1 hybrid seed. Most likely field trials will not be able to start until 2025 to allow for proper characterization of the events and the need to generate adequate seed stocks.
Impacts
What was accomplished under these goals?
Aim 1. Metabolite measurements have been completed. Some potentially informative differences were detected but could not be further studied due to the technology being unavailable because of personnel transitions at the collaborating institution. Rather than spend time seeking an alternative or developing the protocols ourselves, we focused on other aims. Aim 2. All of the transgenic lines envisioned in this aim have been obtained, and the majority of crosses described have been completed. We are currently in the midst of experiments designed to test various aspects of plant performance, as planned. Aim 3. In collaboration with Dr. Long, we will shortly obtain the desired transgenic lines in the LH244 inbred background, which is equally suitable as B73 (APHIS permit is pending).
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Transgenic Expression of Rubisco Accumulation Factor2 and Rubisco Subunits Increases Photosynthesis and Growth in Maize. Kathryn Eshenour, Amber Hotto, Elena J. S. Michel, Zhen Guo Oh, David B. Stern. Under review.
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:Two target audiences (beyond the general scientific community) have been targeted: 1) The interested public. The PD participated in an interactive webinar called "Breaking Ground." This was in a moderator/interview format followed by a Q&A. The session focused on the science behind this NIFA project as well as possibilities for future impact on agriculture. The event was well attended by both a domestic and international audience, and many questions were asked. 2) The private sector. We continue, with assistance of BTI Technology Transfer staff, to seek partnerships to assist in moving our technology to the field. Primarily we have interacted with Corteva and Bayer, since maize is one of their core products and they possess the needed germplasm and technologies. Changes/Problems:The main roadblock has been Aim 3, field trials. We are pursuing multiple solutions and we expect one or more to pan out, but the pace of technology development, or access to it, is to some degree out of our control. We are not alone in facing the difficulty of taking maize transgenics to the field in the public sector. What opportunities for training and professional development has the project provided?Elena Michel, postdoctoral associate, successfully set up radioactive Rubisco activity, content, activation and RCA activity assays, and spectrophotometric ATPase assay. She completed the preliminary data collection for each assay before departing the lab in July. Kathryn Eschenour, Research Assistant, was trained in the various assays established by Elena prior to her departure and is now able to run the in vitro assays independently. Riley Henderson, graduate student, is gaining experience in transgenic maize growth and analysis to determine which events show the most promise for future experiments. She was also trained by Elena to run the in vitro PPDK assays independently. Amber Hotto, Senior Research Associate, was trained by Elena to independently run the radioactive Rubisco activity, content and activation assays. How have the results been disseminated to communities of interest?The "Breaking Ground" webinar/podscast was described earlier. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. The graduate student will attend the Cold Spring Harbor Metabolomics course and be in a position to resume the desired measurements in house. We are hoping to re-invigorate our relationship with the collaborator at Max Planck (post-Covid), however the leadership of that group is retiring and the future availability of the required infrastructure and technical support is unclear. The ability to measure in house will pay off once representative transgenic events are chosen for each transgene and we begin in-depth measurements of performance under the various environmental regimes. Aim 2. This will be the major area of activity as numerous transgenic events for multiple transgenes are characterized in the first and second generations, and crossed the high performing RAF1-LSSS background. Major activities will be growth, transgenic protein accumulation, and in vitro measurements of the relevant metabolic activities. Aim 3. We await the opportunity to create the desired inbred lines and will continue to pursue any and all avenues of potential success.
Impacts
What was accomplished under these goals?
Aim 1.Obtain further insight into improved performance in existing transgenic lines, focusing on the levels of photosynthetic metabolites. 1)Major activities completed / experiments conducted Major activities towards this aim were completed in 2021 and previously reported. 2) Data collected Data was collected and analyzed in 2021 as previously reported. 3) Summary statistics and discussion of results Previous results reported lead us to believe that continued metabolic analysis would give additional insights into photosynthetic carbon flux in transgenic lines to identify potential limitations to photosynthesis and/or areas of increased activity. 4) Key outcomes or other accomplishments realized A new graduate student, Riley Henderson, is poised to develop skills in metabolomics, either at BTI, the Max Planck Institute in Germany, or through a specialized course at Cold Spring Harbor. She applied for the 2022 course and although she was not admitted this year due to pent-up demand, she was encouraged to apply again next year. Aim 2.Create and begin analysis of transgenic maize designed to overexpress RCA or PPDK 1)Major activities completed / experiments conducted RCA Transgenic RCA plants were received from the Iowa State Center for Plant Transformation and confirmed to have either a ZmRCA or At/ZmRCA chimeric transgene by DNA sequencing. Transgenic plants have been grown to the T2 stage and will be ready for thorough analysis soon. Additionally, in vitro radioactive assays have been set up in the lab to measure Rubisco activity, content, activation and RCA activity, as well as a non-radioactive assay to measure ATPase activity of in vitro purified RCA proteins. PPDK Transgenic maize expressing Miscanthus PPDK (MgPPDK) were analyzed in a chilling stress experiment alone, and crossed to previously published RAF1-LSSS lines as planned. Transgenic maize expressing ZmPPDK were screened for PPDK abundance and activity. These plants have been grown to the T2 stage, and events that have increased PPDK abundance and activity will be selected for crossing to RAF1-LSSS for further analysis. An in vitro spectrophotometric PPDK activity assay has been set up in the lab to measure PPDK activity in plant tissue, because transgenic protein accumulation may or may not accurately predict changes in enzyme activity in vivo. A PPDK peptide antibody was made to detect ZmPPDK and MgPPDK with equal affinity for immunoblot quantification. We have made steps to purify the PPDK antibody however this remains a roadblock. 2) Data collected RCA Preliminary data has been collected on the T1 and T2 transgenic maize RCA lines looking at RCA and PPDK protein abundance. Data was collected for three in vitro purified RCA proteins, ZmRCA, At/ZmRCA and AtRCA, comparing their ability to activate Rubisco purified from maize leaf tissue using radioactive Rubisco activity assays. Additionally, one replicate of ATPase assays was completed on the three in vitro purified RCA proteins at temperatures ranging from 25-55?to determine RCA activity at elevated temperatures. PPDK Transgenic maize MgPPDK and MgPPDK-RAF1-LSSS lines were analyzed by measuring photosynthesis, CO2 response, PPDK abundance and PPDK activity under pre-chilling conditions (25°C days / 20°C nights), chilling conditions (14°C days / 12°C nights) and post-chilling conditions (25°C days / 20°C nights). Preliminary data has been collected from the T1 and T2 transgenic maize ZmPPDK lines looking at PPDK and D1 protein abundance. Additionally, the PPDK in vitro assay was used to compare PPDK activities from leaf tissue between ZmPPDK events compared to a WT control. The PPDK peptide antibody was purified using an antigen column and tested via immunoblotting on WT Hi-II plant tissue, maize MgPPDK plant tissue, maize ZmPPDK plant tissue and Miscanthus plant tissue. 3) Summary statistics and discussion of results RCA Preliminary analysis of RCA abundance in T1 and T2 transgenic maize RCA lines indicates that there is no increase in RCA abundance in transgenic lines compared to the WT control. We plan to verify accumulation of transgenic RCA protein accumulation using mass spectrometry in collaboration with BTI faculty Dr. Aleksandra Skirycz who is an expert in this type of analysis. Once transgenic RCA protein accumulation is confirmed, transgenic lines will be assayed under control and heat stress conditions for photosynthesis (using the LI-6800), plant growth and Rubisco activity, for instance. In vitro assays indicate that recombinant ZmRCA, At/ZmRCA and AtRCA are all able to activate maize Rubisco with no significant difference in the activation data. These results confirm that the transgenic RCA in maize will be able to activate maize Rubisco. Preliminary in vitro ATPase assays confirm that At/ZmRCA and AtRCA have higher rates of ATPase activity up to 40 or 45?compared to ZmRCA where ATPase activity starts to decline above 35?. This is as expected. PPDK One transgenic MgPPDK-RAF1-LSS event did not show significant differences in CO2 response or photosynthesis when measured during pre-chilling, chilling and post chilling conditions. Results from the PPDK activity assay and PPDK abundance have not been compiled yet. Additionally, other MgPPDK events and MgPPDK alone could yield different results. Preliminary analysis of PPDK accumulation normalized to D1 protein abundance in T1 and T2 transgenic maize ZmPPDK lines did not show a large difference in PPDK abundance between plant samples. Preliminary in vitro PPDK activity assays of T1 transgenic maize ZmPPDK lines shows certain events have increased ZmPPDK activity compared to both wild-type Hi-II and azygous segregating plants. The PPDK peptide antibody was unable to detect Miscanthus plant tissue PPDK and needs further purification to be used as a primary antibody for this project. Aim 3.Conduct field trials of transgenic lines to validate differences observed under more controlled conditions. Major activities completed / experiments conducted Efforts continue in collaboration with the BTI Center for Biotechnology to achieve in-house maize leaf transformation. We are in discussions with Corteva regarding the ability to field test inbred maize lines that can be generated in their LH244 background. While they are advertising that these lines can be created (at Wisconsin), they are currently evaluating the ability to actually trial the lines on a case by case basis, with no clear guidelines or protocols. We are also continuing collaboration with Univ. Illinois, which is independently attempting to recreate our technology in an inbred maize line that can be field-tested. Data collected None yet. Summary statistics and discussion of results None yet Key outcomes or other accomplishments realized This has been a difficult aim to achieve because the ability to transform inbreds has been overpromised in the public sector, and while the private sector has shared key technologies, the technical support and legal framework are insufficient at present.
Publications
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:We colloquially refer to our project as "turbocharged maize," and as such are able to communicate with non-specialist and non-scientific audiences about our project. We have used the project as an example of the intersection of plant modification and plant performance for the board of directors at BTI, for certain donors to the institute, and as a topic of communication in social media. We also regularly speak with potential industry and foundation partnerswho areinterested in traits that may increase yield in large-scale crops such as maize..although ultimately our strategy may not be limited to maize. Because of Covid there have been very limited opportunities for networking with other plant scientists, at least not in person. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three early-career female scientists have all participated. Elena Michel has been both a trainee and a mentor, shaping her own career but also learning to mentor others who are just beginning to explore the plant sciences. Her mentee, Erin, was a summer REU student who shows incredible potential from both an intellectual and technical point of view. She made a superb public oral presentation, but also achieved important results in the short time she was here. The BTI REU program also has ancillary activities, giving individuals such as Erin a broad view of plant science and its career possibiltiies. She is planning to apply to graduate school this fall. Finally, Kate Eshenour is a "tween" - between undergraduate studies and whatever comes next. She is quickly gaining confidence and independence and truly seems to be enjoying herself. She will continue for another year on the project. Overall, this has been a relatively easy project to explain to its participants and the larger potential impacts are part of the motivation. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will proceed on the three Aims as previously planned, now with the needed personnel and equipment in place. We will primarily be analyzing (rather than creating) transgenic lines, and moving closer to meaningful field work.
Impacts
What was accomplished under these goals?
Impact Optimizing plant performance of commodity plants such as maize, promises to have a tremendous impact on land use, yields and the success of the agricultural enterprise. Our focus on maize may both lead to improvements in field performance, and also suggest paths towards optimization of other crops, particularly C4 crops such as sorghum and sugar cane. This project focuses on improving the assimilation of atmospheric carbon dioxide, which is incorporated into the sugars that drive plant growth. Our goals are improving growth rates and stress tolerance, achieving earlier maturation, gaining yield and as a potential side benefit augmenting carbon sequestration. Accomplishments Aim 1.Obtain further insight into improved performance in existing transgenic lines, focusing on the levels of photosynthetic metabolites. 1) Major activities completed / experiments conducted We completed two rounds of metabolite analysis in conjunction with our collaborators at the Max Planck InstituteinGermany. 2) Data collected Accumulation of a key suite of metabolites relevant to photosynthesis were measured. 3) Summary statistics and discussion of results We anticipated that the increase in photosynthesis in the Raf1-LSSS lines would impact accumulation of photosynthesis metabolites compared to the Hi-II control. Surprisingly, there was not much difference between Calvin-Benson cycle intermediates in the two genotypes, perhaps suggesting that there is no limitation in the carboxylation reaction. There was more aspartate in Raf1-LSSS compared to Hi-II, which could indicate an increase in carbon transport between mesophyll and bundle sheath cells. Additionally, there was a decrease in glycerate, a photorespiratory intermediate, in Raf1-LSSS compared to Hi-II. It is possible that the increase in photosynthesis through an increase in Rubisco carboxylation results in a corresponding decrease in photorespiration. 4) Key outcomes or other accomplishments realized. Research Assistant Angela McEnerney, gained skills in how to use the Licor Li-6800, perform immunoblots, and grow maize plants. Aim 2.Create and begin analysis of new transgenic maize designed to overexpress RCA or PPDK. 1) Major activities completed / experiments conducted; RCA Agave, maize, and the agave/maize chimera RCA constructs were successfully cloned in E. coli and the recombinant proteins affinity-purified. Significant progress has been made towards getting radioactive assays set upto measure Rubisco activity, content, activation, and RCA activity. All three RCA constructs were also transformed into Hi-II maize under the ubiquitin promoter for overexpression. PPDK Maize and Miscanthus PPDKs were introduced into Hi-II maize under the mesophyll-specific PEPC promoter. We have recovered multiple transformantsconfirmed that these plants are expressing PPDK. To better be able to detect and confirm Miscanthus PPDK overexpression in transgenic plants, we have raised a rabbit anti-peptide antibody that is predicted to bind equally to both maize and Miscanthus PPDK. The maize and Miscanthus PPDKs were also cloned with tags into E. coli for subsequent recombinant protein induction and purification. 2) Data collected; RCA project: Three recombinant RCA proteins were purified from E. coli. Preliminary in vitro assays using an older method requiring high amounts of protein were inconsistent, leading us to acquire a specialized spectrophotometer suitable for high-throughput and accurate assays. PPDK project Number of MgPPDK transgenic T0 plants,number of ZmPPDK T0 plants, number of MgPPDK lines shown to have gained the RAF1-LSSS transgene through crossing Preliminary MgPPDK chilling stress trial photosynthesis measurements and growth traits Two recombinant PPDK proteins purified from E. coli. 3) Summary statistics and discussion of results We are assessingwhether Miscanthus PPDK transgenic plants are overexpressing PPDK overall. It is possible albeit unlikely that expression of MgPPDK represses expression of the endogenous PPDK. Miscanthus PPDK and Miscanthus PPDK+RAF1+LSSS T1 plants have a distinct growth phenotype under both control and chilling conditions including a shorter height, and smaller leaf length and area when compared to Hi-II and RAF1+LSSS, as well as a lower maximum carbon assimilation compared to Hi-II and RAF1+LSSS under control growth conditions. These results are preliminary in that only a small number of T1 progeny were the correct genotypes for the experiment, so it will need to be repeated to get enough replicates to calculate statistics. The level of overexpression of PPDK in maize PPDK T0 transgenic plants wasquantified, and there was a range between 1.2- and 1.9-fold overexpression of PPDK in the different events compared to Hi-II. T1 seeds were collected and will be crossed to RAF1+LSSS in the next generation. 4) Key outcomes or other accomplishments realized. Elena Michel, postdoctoral associate, learned how to perform radioactive assays to measure Rubisco activity, content, activation, and RCA activity. Kathryn Eshenour, Research Assistant, gained skills in immunoblotting, recombinant protein induction and purification, and gained expertise performing long-term chilling stress trials on maize. Erin Newringeisen, summer REU intern, learned how to use the Li-Cor and gained skills in immunoblotting, recombinant protein induction and purification, protein activity assays, and gained expertise performing long-term chilling stress trials on maize. Aim 3.Conduct field trials of transgenic lines to validate differences observed under more controlled conditions. This Aim describes moving the maize technology to the field. 1) Major activities completed / experiments conducted; Maize B73 leaf transformation protocols are still being tested at BTI. At the University of Illinois, Stephen Long's lab has created the transformation constructs (RBCS, RBCS-Raf1, and RBCS-Raf1-Raf2) and plan to transform their own maize inbred line for future field trial experiments. 2) Data collected Research specialist Patricia Keenhas been part of an effort joining several transformation facilities to reproduce leaf transformation protocols already in place in industry, with industry collaboration. At stake is a potential breakthrough in accessibility in inbred transformation as well as cost reduction. This is a painstaking effort requiring each step of the protocol to be optimized, however we believe it will be successful at some point during this project. 3) Summary statistics and discussion of results Maize transformation is being set up at the University of Illinois in one inbred line, with a complementary effort at BTI along with its partners. 4) Key outcomes or other accomplishments realized. To troubleshoot maize transformation, Patricia Keen has joined other researchers in a consortium that meets regularly to discuss the technique. They share ideas and problems that they are having with the transformation process, and work together to find a solution. This is not an easy method and others are having similar problems. Having an open network allows them to streamline the troubleshooting process. We are using the legacy Hi-II transformation while we wait but it has disadvantages particularly from a genetic/field trial point of view.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Hotto AM, Salesse-Smith C, Lin M, Busch FA, Simpson I, Stern DB. Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones. J Exp Bot. 2021 Apr 30:erab189. doi: 10.1093/jxb/erab189. Epub ahead of print. PMID: 33928359.
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