Progress 04/15/12 to 04/14/16
Outputs
Target Audience:Springer and Hake have both given seminars at multiple locations for University seminar programs. Both also teach in graduate courses at UC Riverside and UC Berkeley respectively. Springer also teaches an upper division undergraduate course in Plant Anatomy every Fall quarter, using maize leaf mutants for a lab module. Springer gave lectures at a local community college. Hake is involved in outreach and coordinates the USDA garden project that involves 3rd graders from a nearby school and gave one talk to the public on GMOs. Springer runs an annual workshop on GMOs for the UCR Summer REU undergraduate research program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the course of the project, 6 undergraduate students, 5 graduate students, and 2 postdoctoral scientists were trained. Theundergraduates have been trained in basic molecular biology techniques, basic genetics, and plant anatomy. The graduatestudents and postdocs have been trained in advanced genetics and molecular biology, and have provided mentorship to theundergraduates. How have the results been disseminated to communities of interest?Springer and Hake both gave seminars at multiple locations for University seminar programs. Both also taught ingraduate courses at UC Riverside and UC Berkeley respectively. Springer gave lectures at a local community colleges andlectures to students in a Masters program in Biotechnology at the Universidad Autonoma in Madrid. Hake is involved inoutreach. She coordinates the USDA garden project that involves 3rd graders from a nearby school and gave one talk to thepublic on GMOs. Springer gave an annualworkshop on GMOs for the UCR Summer REU undergraduate research program. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Leaves are the major photosynthetic plant organ and are critical for plant productivity. In cereal crops, plants with more upright leaves can be planted at high density, allowing for increased yields. In maize, rice, and other cereal crops, leaf angle depends on the specification and differentiation of two structures, the ligule and auricle, which form at the boundary of sheath and blade. The sheath wraps around the stem and the blade tilts away from the stem to capture light energy for photosynthesis. In maize, a number of mutants alter the formation of the ligule and auricle, and consequently alter leaf angle. Additionally, in maize and rice, mutants that have reduced levels of the plant hormone brassinosteroid (BR) have a more upright leaf than those that accumulate normal levels of the hormone. BRs are a class of hormones that promote growth by regulating cell expansion and cell division. The purpose of this research project was to examine the role of BRs in the development of structures in the maize leaf that impact leaf angle. Our work focused on characterizing the relationship between BRs and genes that are known to regulate aspects of maize leaf patterning. In this project, we demonstrated a direct relationship between the function of the rice transcription factor OSH1 and BR accumulation. This is important because it revealed a new function for OSH1, a transcription factor that functions to regulate identity of the shoot apical meristem, a structure that is critical for plant growth and organ formation. We showed that local reduction of BR accumulation in the blade-sheath junction of rice causes a reduction in leaf angle. This contribution is important because it demonstrated that it is possible to selectively alter BR accumulation and positively impact plant architecture. In contrast, plants that have reduced BRs throughout the plant body have upright leaves, but are dwarf and suffer from low fertility. We generated a number of resources that are important for studying maize leaf development and shoot architecture. These include antibodies that recognize specific proteins that accumulate in the blade-sheath junction. We also identified genes that are expressed specifically in the auricle, which will be important markers for studying leaf development. We characterized transgenic maize plants that have reduced BR perception and characterized several additional maize mutants that have connections to BR pathways. We also identified genes that are markers of different domains in the maize leaf. Our specific accomplishments are as follows. We demonstrated that in rice, the KNOX transcription factor OSH1 directly activates transcription of BR catabolic enzyme genes CYP734A2 and CYP734A4. This activity serves to limit the accumulation of BRs in the meristem. In transgenic rice expressing the Arabidopsis BR-catabolic gene BAS1 under control of a boundary-specific promoter, we documented specific effects on leaf angle and panicle architecture. Intriguingly, these transgenic plants had more erect leaves, increased tassel branches, and were statistically taller than control plants. This suggests that plants compensate for reduction of brassinosteroid in local regions by increasing growth in adjacent regions. We documented leaf patterning and tassel architecture phenotypes in maize transgenic plants that have reduced expression of the BR receptor BRI1 or the BR signaling gene BIN2. We introgressed the BRI1 and BIN2 RNAi lines into a variety of mutant backgrounds to assess the impact of altered brassinosteroid signaling on leaf patterning. Analysis of the resulting phenotypes is still ongoing. We also characterized some new mutants that alter maize leaf patterning and investigated their relationship with known components of BR signaling and maize leaf development. Unfortunately, our efforts to develop virus induced gene silencing (VIGS) as a method for silencing genes in the maize leaf have been unsuccessful. We tested 15 different inbred lines as well as several mutant backgrounds that we thought might be more susceptible to virus infections. We occasionally observed symptoms that suggested the virus was replicating, but we were not able to detect viral transcripts. We therefore conclude that this version of the barley stripe mosaic virus (BSMV) is not effective for VIGS in maize, despite reports that it has been used successfully (Holzberg et al. 2002).
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Tsuda K and Hake S (2016) Homeobox transcription factors and the regulation of meristem development and maintenance. In Plant Transcription Factors: Evolutionary, Structural and Functional Aspects
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Tsuda K and Hake S (2015) Diverse functions of KNOX transcription factors in the diploid body plan of plants. Curr Opin Plant Biol 27, 91-96.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Sluis A, and Hake S (2015) Organogenesis in plants: initiation and elaboration of leaves. Trends Genet 31, 300-306.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Diaz, J (2016) Characterization of the Role of LOB-DOMAIN Genes and Brassinosteroids
in Rice Architecture. In Plant Biol, Ph.D. (Riverside: University of California), P. 192.
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Progress 04/15/14 to 04/14/15
Outputs
Target Audience:Springer and Hake haveboth given seminars at multiple locations for University seminar programs. Both also teach in graduate courses at UC Riverside and UC Berkeley respectively and Springer teaches an upper division undergraduate course inPlant Anatomy. Hake is involved in outreach, coordinating theUSDA garden project that involves third graders from a nearby school and gave two talks to thepublic on GMOs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for 3 undergraduate students, 5 graduate students, and 2 postdoctoral scientists. The undergraduates have been trained in basic molecular biology techniques, basic genetics, and plant anatomy. The graduate students and postdocs have been trained in advanced genetics and molecular biology, and have provided mentorship to the undergraduates. How have the results been disseminated to communities of interest?Hake gave seminars atUC Riverside (IIGB/CEPCEB Student-Invited Seminar, 9/14/2014);University of Missouri (Interdisciplinary Plant Group,2/17/2015);Monsanto in St. Charles, Missouri (3/11/2015);University of Illinois (IGB Pioneers Seminar,4/14/2015). Springer gave a seminar at UC Riverside (REU summer undergraduate program, 6/14/2015). What do you plan to do during the next reporting period to accomplish the goals?Our efforts will focus on characterization of the transgenic Bin2-RNAi and Bri1-RNAi plants in various mutant backgrounds and conducting RNA-seq in these backgrounds. We will test other BSMV isolates for their ability to infect maize leaves.
Impacts
What was accomplished under these goals?
Feminised upright narrow (fun1) is a pleiotropic mutant with sex determination and leaf architecture defects. The tassel is feminized and the adult leaves are narrow and lack auricles giving them an upright habit. Both of these defects are reminiscent of brassinosteroid defects; nana mutants, which are defective in brassinosteroid biosynthesis are feminized (Hartwig et al. 2011) and Bri1-RNAi plants, which reduce levels of the brassinosteroid receptor, have reduced auricles (Kir et al. 2015). We cloned fun1 by map based cloning followed by RNAseq and found one mutation in the interval of 10 genes. This allele came out of a B73 EMS mutant screen. We identified another mutant in an A619 EMS screen and found the two mutations did not complement. Both mutations lead to stop codons. The fun1 gene encodes a protein of unknown function that is conserved in the grasses. One domain is conserved across the Plant Kingdom. We now have an antibody to determine its cellular and tissue localization. Double mutants have been made to place fun1 in a genetic pathway. Wab1-R is a dominant mutation that overexpresses a TCP transcription factor. This overexpression leads to ectopic LG1 accumulation (Lewis et al. 2014). The Wab1-R;fun1 double mutant displays a synergistic phenotype with a novel leaf structure, implying that these proteins may converge on the same pathway. Leaves are very narrow and shorter, similar to Wab1-R; lg1 double mutant leaves. This result suggests that FUN may be involved in LG1 accumulation or that Wab1-R upregulates FUN. Silkless (sk1) is a classical maize mutant that fails to make silks in the ear. The silkless1;fun1 double mutant has loss of silks in both ears and tassels, but has a synergistic phenotype in the tassel in which branches fail to initiate. This result is intriguing and suggests FUN may play a role in tassel branch initiation. D8 and D9 are mutations in the DELLA domain of nuclear-localized negative regulators of gibberellin signaling. The dominant D8 and D9 mutants are dwarfs with masculinized ears. D8;fun1 double mutants looks like the D8 single mutant implying that D8 is epistatic. These results suggest that GA may be involved in the fun1 pathway. Another mutant we have recently started to analyze is narrow odd dwarf1 (nod1). nod1 encodes the maize ortholog of MCA1 (MID COMPLEMENTING ACTIVITY 1). MCAs are land plant-specific, broadly expressed and membrane-localized proteins with suggested calcium channel-related activity (Nakagawa et al. 2007). The nod1 phenotype is very severe in B73 with small, liguleless leaves and a loss of apical dominance. Cell divisions and differentiation are erratic. We carried out an RNAseq experiment, but found 4000 differentially expressed genes, clearly too many to make specific hypotheses. Now we are using an antibody to immunoprecipitate NOD1-associated proteins. In our first experiment we found two proteins (GRMZM2G107444, a membrane steroid-binding protein 1 and GRMZM2G126019, a dynamin-related protein 1C) that are implicated with BR signaling. Future experiments will confirm this result and identify other interactors. Interestingly, although the ligule region is very abnormal with unorganized divisions and a total failure to produce a ligule or auricles, LG1 accumulation is normal. This result suggests that NOD functions downstream of LG1, in the elaboration of ligule/auricle production. We have also continued to characterize transgenic maize lines that have reduced brassinosteroid responses. Because of the need to introgress the transgenic maize lines into standard inbred backgrounds before phenotypic characterization, this objective has taken longer than anticipated. The transgenic lines have now been back-crossed several times and we will be able to obtain detailed phenotypic data. Our efforts to develop VIGS as a method for silencing genes in the maize leaf have been unsuccessful. We tested 15 different inbred lines as well as several mutant backgrounds that we thought might be more susceptible to virus infections. We occasionally observed symptoms that suggested the virus was replicating, but we were not able to detect viral transcripts. We therefore conclude that this version of the barley stripe mosaic virus (BSMV) is not effective for VIGS in maize, despite reports that it has been used successfully (Holzberg et al. 2002). We will test other BSMV isolates for their ability to infect maize leaves. Hartwig T et al (2011) Brassinosteroid control of sex determination in maize. Proc Natl Acad Sci USA 108:19814. Holzberg S et al (2002) Plant J 30:315. Kir G et al (2015) Plant Physiol 169:826. Lewis MW et al (2014) Development 141:4590. Nakagawa Y et al (2007) Proc Natl Acad Sci USA 104:3639.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Tsuda K, Kurata N, Ohyanagi H, and Hake S (2014) Genome-wide study of KNOX regulatory network reveals brassinosteroid catabolic genes important for shoot meristem function in rice. Plant Cell 26, 3488-3500.
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Progress 04/15/13 to 04/14/14
Outputs
Target Audience:Springer gave seminars at multiple locations for University seminar programs. Springer and Hake each teach in graduate courses at UC Riverside and UC Berkeley respectively. Springer also taughta newupper division undergraduate course in Plant Anatomy, withmaize leaf mutants used for a lab module. Hake is involved in outreach, coordinating the USDA garden project that involves third graders from a nearby school and gave two talks to the public on GMOs. Springer gave a lecture to the summer undergraduate REU program, and ran a workshop on GMOs for the REU students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for 2 undergraduate students, 3 graduate students, and 2 postdoctoral scientists. The undergraduates have been trained in basic molecular biology techniques, basic genetics, and plant anatomy. The graduate students and postdocs have been trained in advanced genetics and molecular biology, and have provided mentorship to the undergraduates. How have the results been disseminated to communities of interest?Springer gave lectures at Centro Nacional de Biotecnología (CNB), Madrid, Spain, "Hormones and boundaries regulate shoot architecture",April 2013;Sainsbury Laboratory, Cambridge, England "Life on the edge: transcription factors that regulate organ boundaries", April 2013; UC Riverside Center for Plant Cell Biology Symposium, "Organ boundaries and hormones regulate plant architecture", Dec 2013. What do you plan to do during the next reporting period to accomplish the goals?We are optimizing conditions for treatment with exogenousBrassinosteroids and the BR-biosynthesis inhibitor and are testing the transcriptional response of BR-rsponse genes. We then plan to do RNA-seq on dissected blade, auricle, and sheath following chemical treatments to identify BR-response genes in discrete regions of the leaf. We will continue to quantify phenotypes resulting fromBRI1-RNAi and BIN2-RNAi in various genetic backgrounds. We will continue to test VIGS in the maize leaf. We have collected a set of diverse maize inbreds and are testing different infection methods, including vacuum infiltration,introduction by abrasion, and leaf puncture. Our goals are to silence genes in the BR pathway to assess the impact on maize leaf patterning.
Impacts
What was accomplished under these goals?
The goals of this project are to develop an understanding of how BR signaling impacts maize leaf patterning. Our first objective aims to describe the leaf phenotypes resulting from increased or decreased BR signaling. We are characterizing BR biosynthesis mutants, transgenic maize plants that have increased or decreased BR response, and maize plants that are treated with exogenous BR or BR biosynthesis inhibitors. We obtained two transgenic lines from Phil Becraft, Bri1-RNAi (reduced BR signaling) and Bin2-RNAi (increased BR signaling). In Bri1-RNAi plants the auricles appear smaller and the boundary between the auricle and the blade is diffuse, whereas in Bin2-RNAi plants, auricles are larger. We also noted changes in tassel branch number and angle, indicating a role for BR in these aspects of inflorescence architecture. We crossed Bin2-RNAi and Bri1-RNAi to several maize mutants that have altered leaf patterning, including gain of function knox mutants and Wavy auricle in blade1 (Wab), which both make auricles in the blade, and liguleless1 (lg1), which lacks a ligule/auricle and has more upright leaves. These studies have revealed that Liguleless1 activity is required for the BIN2-RNAi phenotype, suggesting lg1 acts downstream of BR signaling. In contrast, Bin2-RNAi partially suppressed the Wab phenotype, suggesting the BR signaling is reduced in Wab mutants. Our second objective is to identify genes that function in BR signaling in discrete tissues of the maize leaf. Toward this goal, we have conducted RNA-seq experiments on RNA isolated from dissected blade, auricle, and sheath tissues. These experiments have identified a number of candidate marker genes for each of the tissues, which will be allow more precise phenotypic analyses of mutants. We are optimizing conditions for BR and inhibitor treatments and will repeat the RNA-seq experiments on tissues from treated plants in the coming months. We have also characterized a BR-catabolic enzyme in rice that is a target of the rice knox1 transcription factor OSH1 and demonstrated a role for this gene in leaf angle. We are also developing VIGS as a method to silence genes in the maize leaf. We have generated a series of VIGS constructs using Barley Stripe Mosaic Virus (BSMV). We are testing a variety of maize inbreds for infection.
Publications
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Progress 04/15/12 to 04/14/13
Outputs
Target Audience:Springer and Hake have both given seminars at multiple locations for University seminar programs. Both also teach in graduate courses at UC Riverside and UC Berkeley respectively. Springer gave lectures at a local community colleges and twolecture to students in a Masters program in Biotechnology at the Universidad Autonoma in Madrid..Hake is involved in outreach. Shecoordinatesthe USDA garden project that involves 3rdgraders from a nearby school and gave one talkto the public on GMOs. Springer gave a workshop on GMOs for the UCR Summer REU undergraduate research program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for 3 undergraduate students, 3 graduate students, and 2 postdoctoral scientists. How have the results been disseminated to communities of interest?Hake gave a seminaratThe John Innes Centre:The Biffen Lecture, "Patterning the maize leaf", 9/14/2012. Springer gave a seminarat Kansas State University Department of Biology ,"Life on the Edge - Organ Boundaries Regulate Shoot Architecture"3/15/2013 and a seminar at UC Riverside, summerundergraduate REU program, 6/2012). What do you plan to do during the next reporting period to accomplish the goals?Activities in the next project period will focus on characterizing the phenotypic effects of altered BR signaling and response in maize leaves, using treatment with the BR-biosynthesis inhibitor propiconazole, and characterization of the BRI1-RNAi and BIN2-RNAi plants. We will also test VIGS for gene silencing in maize, using the constructs we have generated.
Impacts
What was accomplished under these goals?
The maize homeodomain transcription factor KNOTTED1 (KN1) and OSH1, the rice ortholog, target genes in the brassinosteroid pathway. Treatment of the Kn1-N dominant gain-of-function mutant with propiconazole, an inhibitor of BR synthesis, enhances the phenotype, resulting in plants with sheath transformations along the veins and a lack of ligule. This result suggests that KN1 functions to reduce BR and that by further reducing BR, we accentuate the phenotype. We carried out a Dexamethasone-induction experiment using OSH1-GR to determine which BR genes are bound and modulated by OSH1. We found that BR catabolic enzyme genes CYP734A2 and CYP734A4 were induced in 3 hours. In osh1 loss-of-function mutants, their expression level was reduced. Chromatin immunoprecipitation assays showed that both loci were bound by OSH1 in wild-type shoot apices. In fitting with the BR levels, the leaf-bending angle was greater in osh1 mutants than in wild-type, and smaller in DEX-treated OSH1-GR plants. We also discovered that the short sheath phenotype observed in DEX-treated OSH1-GR plants were attenuated by BR treatment. To explore the relationship of knox genes and BR in the inflorescence, double mutants were made with nana1, a mutation in a biosynthetic step of BR synthesis (Hartwig et al. 2011), and kn1-e1, a loss of function allele. kn1-e1 tassels are sparse, with fewer branches and fewer spikelet pairs. In our field and greenhouse conditions, the nana mutants had normal tassels. Out of 7 double mutants scored in the field, 2 were tasselless, 2 were tasselseed and 3 were normal (but smaller than their non-mutant siblings). In a related family grown in the greenhouse, 2 were tasselless, 3 were tasselseed and 2 normal. If KN1 simply reduced BR, then loss of BR might compensate for the loss of KN1 and the phenotype restored to normal. These results demonstrate a synergy with the loss of kn1 and reduced BR levels. Thus, it is likely that KN1 functions in the leaf differently from how it functions in the meristem. We made antibodies to both Liguleless1 (LG1) and LG2. The LG1 antibody has been very useful. LG1 is expressed at the preligule region prior or just as divisions start. It is also expressed in the axil of tassel branches. This expression pattern is consistent with the fact that lg1 mutants are not only missing ligules but have more upright branches. We obtained two transgenic lines from Phil Becraft, Bri1-RNAi and Bin2-RNAi. Bri1-RNAi plants look very similar to KNOX dominant mutants, with an extension of sheath cells into the blade and reduced auricles. In contrast, Bin2-RNAi auricles are larger. We noticed that the Bin2-RNAi tassel branches are longer and more outward. This discovery fits with the correlation of tassel branch angle and leaf angle we have seen with lg1, lg2, Lgn and Wab. We crossed Bin2-RNAi to both Mo17 and B73 and are growing larger families in the field this summer. We will carry out histology of the auricle and tassel branch angle as well as LG1 immunolocalizations on both Bri1-RNAi and Bin2-RNAi lines this summer. We cloned Lgn, which encodes a kinase. As a control for kinase activity, we have used the Arabidopsis BRI1 protein. BRI1 phosphorylates LG1, but not LG2 nor LGN. Interestingly, the phosphorylation activity of BRI1 decreases in the presence of LG2. We have cloned the maize version of BRI1 and see the same result. Our colleagues on an NSF funded project have carried out laser capture of the preligule band and just above and below the ligule region. We have examined this gene list for BR genes, and interestingly, several BR-signaling genes are enriched in the preligule region. We are working to develop virus-induced gene silencing (VIGS) as a technique to silence genes in the maize leaf. We obtained Barley Stripe Mosaic Virus (BSMV) VIGS vectors from Andy Jackson (Yuan et al, 2011) and have cloned lg1 and the control gene phytoene desaturase (PDS1) into these vectors. We are currently infecting Nicotiana benthamiana with these to obtain innoculm for use in maize infections.
Publications
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