REGULATION OF LEAF GROWTH SHAPES AND VEIN PATTERNS IN BIRCH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1008782
• Project Start Date: Nov 13, 2015
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN TECHNOLOGICAL UNIV
1400 Townsend Drive
HOUGHTON,MI 49931

Performing Department
College of Forest Resources and Environmental Science

Non Technical Summary
As the main photosynthetic sites of vascular plants, leaves play essential roles in providing carbohydrates for plant growth and development. Despite this importance, the molecular mechanisms underpinning leaf growth in plants have not been revealed. Currently, the completion of birch genome sequences, the availability of high-throughput gene expression profiling methods, and the developed state-of-the-art gene network construction methods have made it an opportune time to initialize systems biology research to accomplish this goal. Here, we propose to construct the gene regulatory network governing leaf growth shapes, and vein patterns in birch. To accomplish this, we have generated gene expression data sets and built a gene regulatory network for identifying the critical regulators. Assuming that the genes that are linked to existing known leaf regulators in the built network are functionally associated, we will select these genes to test through transgenic approach. We will examine if overexpression/ suppression of these selected genes can cause significant changes in leaf growth shapes, sizes, and vein patterns.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

100%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	0699	1040	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0699 - Trees, forests, and forest products, general;

Field Of Science
1040 - Molecular biology;

Keywords

leaf growth shape

size

vein pattern

gene regulatory network

and transgenic line

Goals / Objectives
We aim to identify high hierarchical regulators controlling leaf growth in the bioenergy tree crop, Betula pendula, and then experimentally validate their roles in leaf growth and development for developing a novel model system to reliably predict phenotype from genotype. Specifically, we have the following objectives:To identify new regulators that control leaf growth shapes, sizes and vein patterns.To produce overexpression or suppression transgenic lines, and then evaluate the roles of selected genes that may possibly govern leaf growth shapes and vein patterns

Project Methods
Materials: European white birch, Betula pendula, has different shapes of leaves and vein patterns (Fig. 1). We crossed two B. pendula trees that have unlobed leaves, and obtained some offspring with moderately or deeply lobed leaves, as shown in Fig. 1. The ratio of lobed to unlobed leaves in offspring indicated that there are a few loci that control the leaf shapes and the vein patterns. In this study, we used trees with moderately lobed leaves for experimental materials and trees with unlobed leaves as the controls, and performed an comparative RNA-seq experiment to generate transcriptomic gene expression data for identifying differentially expressed genes and building a gene regulatory network.MethodsIdentification of genes controlling leaf growth shapes, sizes, and vein patterns through network constructionWe have generated gene expression profiles during leaf development. The data from six developmental stages were analyzed to identify differentially expressed genes (DEGs) at each of the developmental stages. Using these DEGs and transcription factors as inputs for the ARACNE, an algorithm for the reconstruction of gene regulatory networks, we built the gene regulatory network for birch (Fig. 3), from which we identified a number of candidate genes (highlighted in green) for experimental analysis. We are cloning these genes for developing overexpression and suppression transgenic lines to test if they play a role in leaf development.Generation of transgenic lines: To generate over-expression transgenic lines, we will amplify the full-length of each selected regulatory gene present in the gene regulatory network, and clone it into the pCAMBIA1300 vector. It will be driven by the 35S promoter. To generate suppression lines via RNAi technology, we will use the PK7GWIWG2(II) (http://www.vib.be/VIB/EN/) expression binary vector that was modified from a small binary vector called pPZP222 for generating efficient RNAi constructs. Gene-specific fragments of 200~300 bp will be cloned into the PK7GWIWG2(II) vector at two different loci and in opposite orientation to facilitate the formation of double-strand RNAs that are extraordinarily efficient in triggering gene silencing. The constructs will be transferred into the Agrobacterium tumefaciens AGL1 strain using a freeze-thaw protocol (Holsters, de WD et al. 1978). To generate transgenic lines, we will use the A. tumefaciens containing different constructs to infect the leaves of white birch with our standard transformation procedure. We will generate at least 5 independent transgenic lines per gene construct for selecting up/down-regulated lines with various efficiencies.Characterization of transgenic plants: All transgenic plants will be propagated in vitro and transferred to a greenhouse and grown under regular conditions. Their growth will be monitored and compared to wild-type plants. Transgenics will be subjected to molecular analyses to characterize the integration of transgene and level of transgene expression using RT-PCR (normalized to ubiquitin/cyclophilin) in order to select the desirable lines for further characterization. Leaf growth will be measured daily (AB Electonics Ltd, Romford, Essex, UK) as described in (Morabito, Caruso et al. 2006). Analysis of leaf surface area will be carried out with Image software. The significantly altered transgenic lines will be subjected to more molecular characterization for publication purposes.

Progress 11/13/15 to 09/30/19

Outputs
Target Audience:(1) Tree biotechnology research community; (2) Forest owners including forest companies and corporations; (3) Research sponsors; Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. An opportunity is provided to an undergraduate students, Joshua Marshall, in Department of Biological Science, 2. An opportunity is provided to a graduate students, Jianlin Lei, in Department of Computer Science, 3. An opportunity is provided to an undergraduate student, Tyler Ollanketo, n Department of Biological Science, 2. An opportunity was provided to a tempory employee, Yinan Yuan, who is an Research Assistant Professor in School of Forest Resources and Environmental Science How have the results been disseminated to communities of interest?The project is still ongoing and we have not disseminated the result to the research community. What do you plan to do during the next reporting period to accomplish the goals?Some revisions of the manuscript will be done in next year. This will be done at no additional cost.We will then try to submit it.

Impacts
What was accomplished under these goals? (1) We generated gene expression data from birch leaves of different developmental stages; (2) We built a gene regulatory network to identify the genes that potentially control leaf development and size, and a homeodomain box transcription factor was identified and was named HD4; (3) We characterized its tissue specific expression pattern using PCR and we found its expression gradually increases as leaves become older; (4) We isolated HD4 gene from birch genome. (4) We fused the coding region of HD4 gene with 35 S gene promoter to generate an expression cassette and placed it into the pCAMBIA1300 binary plasmid vector. (5) We developd the transgenic lines and screened the transgenic lines with qRT-PCR, (6) We measured the growth of HD4 trangenic lines including leaf wide, leaf length, and area. (7) We identified some binding motifs of HD4 gene using TF-cented Y1H and yeast one-hybrid (Y1H) experimental system, and fould that HD4 binds to two DNA motifs: (a) GTGANTG10 motif: TTGATGTGAT; (2) LTRECOREATCOR15 motif:CCGACGCCTG; (3) ARR1AT motif: TTGATTTGA; (4) CGACGOSAMY3 motif: TCGACGCGT

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ji, X. S. Chen, J. Li, W. Deng, Z. Wei,and H. Wei*. 2017. SSGA and MSGA: two seed-growing algorithms for constructing collaborative subnetworks. Scientific Reports. 7:1446, DOI:10.1038/s41598-017-01556-z. We have a manuscript which still needs some minor work before it can be considered for publishing.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Gunasekara, C, K. Zhang, W. Deng, L. Brown and H. Wei*. 2018. TGMI: an efficient algorithm for identifying pathway regulators through evaluation of triple-gene mutual interaction. Nucleic Acids Res. 46(11):e67.
• Type: Other Status: Other Year Published: 2020 Citation: A homeodomain box 4 gene controls birch leaf size (in preparation)

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:1. Tree biotechnology research community 2. Forest owners, nurseries and corporations 3. Governmental research sponsors 4. College students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The opportunity is provided to undergraduate student, Tyler Ollanketo, graduate student, Chathura Gunasekara,and a scientist, Yinan Yuan. How have the results been disseminated to communities of interest?The project is ongoing and we are still organizing the results. We do not have anything to report about dissemination at this time. What do you plan to do during the next reporting period to accomplish the goals? We will try to organize the results and try to publish the manuscript next year.

Impacts
What was accomplished under these goals? In the past year, we grew the sixoverexpression transgenics lines of homeodomain 4 gene (HD4), a leaf-size regulator we identified, and performed RNA-seq experiment to identify itscandidate target genes. First, the expression data (RNA-seq data) wereanalyzed to identify differentially expressed genes thatresponedto the HD4's overexpression, we then performed pathway and gene ontology enrichmented analysis to identify the other biological processes that were impacted by HD4 overexpression. Using the gene expression profiles of HD4 and its responsive genes, webuilt a HD4-mediated hierarchical network. We also generated antisense transgenic lines to investigatethe impact of leaf sizeswhenthe HD4's expression wastuned down. The plants are growing for further analysis. Table 1: The potential targets of Homeodomain HD4 genes ID_birch Homologs in Arabidopsis Short_description Bpev01.c1653.g0004.m0001 AT4G03270.1 Cyclin D6;1 Bpev01.c1152.g0009.m0001 AT3G10330.1 Cyclin-like family protein Bpev01.c1213.g0001.m0001 AT3G20860.1 NIMA-related kinase 5 Bpev01.c0294.g0003.m0001 AT5G42080.2 dynamin-like protein Bpev01.c0294.g0006.m0001 AT3G14440.1 nine-cis-epoxycarotenoid dioxygenase 3 Bpev01.c0162.g0039.m0001 AT4G23230.1 cysteine-rich RLK (RECEPTOR-like protein kinase) 15 Bpev01.c1513.g0006.m0001 AT1G24430.1 HXXXD-type acyl-transferase family protein Bpev01.c2531.g0001.m0001 AT5G02500.1 heat shock cognate protein 70-1 Bpev01.c0490.g0001.m0001 AT4G21390.1 S-locus lectin protein kinase family protein Bpev01.c0870.g0008.m0001 AT4G19230.1 cytochrome P450, family 707, subfamily A, polypeptide 1 Bpev01.c0353.g0015.m0001 AT1G15550.1 gibberellin 3-oxidase 1 Bpev01.c1670.g0008.m0001 AT4G27410.2 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein Bpev01.c0781.g0009.m0001 AT1G65910.1 NAC domain containing protein 28 Bpev01.c0123.g0010.m0001 AT5G19820.1 ARM repeat superfamily protein Bpev01.c0171.g0040.m0001 AT4G27410.2 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein Bpev01.c0397.g0016.m0001 AT3G07040.1 NB-ARC domain-containing disease resistance protein Bpev01.c0597.g0002.m0001 AT2G36230.1 Aldolase-type TIM barrel family protein Bpev01.c0088.g0098.m0001 AT1G01720.1 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein Bpev01.c0480.g0058.m0001 AT1G20930.1 cyclin-dependent kinase B2;2 Bpev01.c0268.g0003.m0001 AT4G19170.1 nine-cis-epoxycarotenoid dioxygenase 4 Bpev01.c0594.g0012.m0001 AT1G22710.1 sucrose-proton symporter 2 Bpev01.c0016.g0150.m0001 AT4G15550.1 indole-3-acetate beta-D-glucosyltransferase Bpev01.c0016.g0149.m0001 AT4G15550.1 indole-3-acetate beta-D-glucosyltransferase Bpev01.c0823.g0006.m0001 AT1G17120.1 cationic amino acid transporter 8 Bpev01.c0051.g0021.m0001 AT5G56840.1 myb-like transcription factor family protein Bpev01.c1907.g0007.m0001 AT1G17060.1 cytochrome p450 72c1 Bpev01.c0298.g0008.m0001 AT5G23940.1 HXXXD-type acyl-transferase family protein

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Gunasekara, C, K. Zhang, W. Deng, L. Brown and H. Wei*. 2018. TGMI: an efficient algorithm for identifying pathway regulators through evaluation of triple-gene mutual interaction. Nucleic Acids Res. 46(11):e67. (IF = 11.53)
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wei, H. 2017. Construction of a hierarchical gene regulatory network centered around a transcription factor. Briefing in Bioinformatics. DOI:10.1093/bib/bbx152. (IF=7.3)

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience of this project includes: 1. Tree biotechnology research community 2. Forest owners, nuseriesand corporations 3. Governmental research sponsors 4. College students Changes/Problems:We did not encounter any problem and nothing has been changed. What opportunities for training and professional development has the project provided?The opportunitieswereprovided to Joshua Marshell, a graduate student in biology, and Jialin Lei,a graduate studentin computer science. How have the results been disseminated to communities of interest?The project is ongoing and we are still organizing the results. We do not have anything to report about dissermination at this time. What do you plan to do during the next reporting period to accomplish the goals?We are organizing amanuscript and willtryto publish itduring the next reporting period though we still need to do some lab work. 1. We will measure if the contents of some hormones including auxin,GA andABAin different positions of leaves have changed. This may help usinterpret why overexpression of HD4 gene can cause theleaf expansion. 2. We will try to do some experments to identify the true target genes of HD4, which will further help us to expainwhy overexpression of HD4 gene can cause theleaf expansion. 3. Organize the results and publish the manuscript.

Impacts
What was accomplished under these goals? Summary of the progress: In the past year, we identified a homeodomain (HD) 4 gene(HD4)from the hierarchical genesnetwork we built usingbirch high-throughout gene expression data, and then transformed the gene into thebirch genome using Agribacterium. We generated 6transgenic lines, and the plants have been grown in pots.We investigated the growth characteristics of these transgenic lines and obtaind the following data: Transgenic Lines Tree Height (cm) Leaf Width (cm) Leaf Length (cm) Leaf Areas (cm2) ------------------------------------------------------------------------------------------------------------------------------------------------------- Controls 45.0 3.96 5.33 14.10 L41 36.3 4.17 5.87 16.30 L43 31.6 4.50 5.83 15.50 L410 52.7 5.03 6.60 14.49 L414 45.7 5.13 5.93 14.73 L416 51.3 4.73 6.26 20.77 L419 44.3 6.23 7.20 22.92 ---------------------------------------------------------------------------------------------------------------------------------------------------------------- * The data shown are the averages from five replicates. Although we have not performedstatistical analysiss and multiple comparisons on these data, the evidence shown above clearlysupports the hypothesis that HD4plays a role in increasing both leaf lengthand leaf width, resulting in the increased leafareas. However, no consistent effects of HD4 on height growth isobserved. Considering the plants are still young, we are planning tonvestigate these growth characteristicsagain next year when the plants are bigger andrelatively well-developed. We also spent time reanalyzing the RNA-seq data we obtained after the silver birch genome was published by Finland's group in2017. We realigned our sequence readsto silver birch genome andgenerated the raw counts(expression levels) for all genes. Wehave not builtthe hierarchical gene regulatory network but will do this in next reporting period to obtainthe network topology and context of this HD4 gene, which may help us to understand HD4'sfunctions in promoting leaf growth.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ji, X. S. Chen, J. Li, W. Deng, Z. Wei,and H. Wei*. 2017. SSGA and MSGA: two seed-growing algorithms for constructing collaborative subnetworks. Scientific Reports. 7:1446, DOI:10.1038/s41598-017-01556-z (IF=5.3).
• Type: Journal Articles Status: Other Year Published: 2018 Citation: A homeodomain box 4 gene controls birch leaf size (in preparation for submission)

Progress 11/13/15 to 09/30/16

Outputs
Target Audience:Target audience of this project: Tree biotechnology research community Forest owners, including corporations Governmental research sponsors Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An opportunity was provided to an undergraduate student, Joshua Marshall, in the Biological Sciences Department at Michigan Tech. He worked on this project for 3 months in the summar of 2016. How have the results been disseminated to communities of interest?The project is still ongoing and we do not have anything to report with regard to the dissemination of results at this time. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will do the following tasks: 1. We will fuse the coding region of HD4 gene with 35 S gene promoter to produce an expression cassette. 2. We wil place this expression cassette into the pCAMBIA1300 binary plasmid vector, where the 35 S promoter can activate and drive the HD4 gene to express itself at high level when the binary vector harboring 35 S-HD4 expression cassette is transformed and integrated into plant genome. 3. We will deliver the above binary vector horboring the 35S-HD4 expression cassette into poplar or birch genomes. 4. We will develop the transgenic lines and examine the alterations in leaf growth and leaf sizes. Our long-term goal is to identify and characterize a gene that can increase leaf growth and expansion of plant species especially trees, and the ultimate goal is to increase wood productivity in trees by enhancing the photosynthesis capacity.

Impacts
What was accomplished under these goals? Summary of the progress: In the past year, we isolated a Homeo-domain 4 gene (HD4, original ID CCG009086.1) and then characterized the tissue-specific expression pattern of this gene in multiple tissues of birch seedlings, which include apical meristem (top/tip), and the first leaf (tiny, unexpanded) through the sixth (fully expanded) leaf. We found HD4 is differentially expressed in various developmental stages of leaves. The expression levels of HD4 in the apical meristem and the growing leaves are : Meristem 194.3 First leaf 469.3 Second leaf 696.3 Third leaf 1254.4 Fourth leaf 2987.7 fifth leaf 3523.5 Six leaf 4114.2 (All expression values shown above were calculated from three biological replicates) The expression of this gene was relatively low in the apical meristem. If we set HD4's expression level in the apical meristem as a baseline, its expression levels in first, second, third, fourth, fifth and sixth leaves were 2.4, 3.6, 6.5, 15.4, 18.1, and 21.2 times more than that in the apical meristem, respectively, suggesting HD4 may play a role in leaf growth and expansion. HD4's homologous gene in Arabidopsis thaliana is MYB91 (AS1), which is knwon to play a critical role in the expanding leaves through controling the proximal-distal symmetry determination. AS1 inArabidopsis thaliana is expressed at a higher level at leaf tips than anywhere esle in leaves during leaf expansion. To examine if HD4 from birch is also expressed at a higher level at the leaf tips of birch, we measured its expression at the leaf tips in comparison with the middle areas of leaves. We found the expression of HD4 at the leaf tips was indeed at a higher level compared to the middle regions of leaves. HD4 expression levels : Leaf tips: 1309 Central area of leaves: 896 (the values were averaged from three replicates) The evidence shown above supports the hypothesis that HD4 may regulate leaf growth and expansion. If this hypothesis is true, overexpression of this gene in tree species may potentially increase leaf size.

Publications