Progress 10/01/13 to 09/30/18
Outputs
Target Audience: Target audiences included undergraduate students (>30) enrolled in the PI's courses, PLSC 4210 Plant Physiology, How Plants Work and high school and undergraduate students through guest lectures as well as scientist and researchers that attended the ASPB 2018 meeting and Plant Biology symposium 2018 at UMass. Readers of our published paper resulting from this project were also our target audience. The PI was invited to give guest lectures for high school students at Woodstock Academy on Oct 8, 2017, and for Edwin O Smiths High School on May 2nd, 2018. The PI also lectured for the Sustainability 360 class at UConn on Mar 7, 2018. Students attending the PI's lectures for the course and guest lectures were provided with information about biofuel production and environmental impact of fossil fuels, and the regulation of vascular development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training opportunities were provided to graduate students and undergraduate students. One graduate student, Qian Du, worked on investigating gene functions in controlling vascular development. She was trained in experimental design and various molecular techniques. Three undergraduate students participated in the project by helping to care for plants, collecting materials, and performing experiments. How have the results been disseminated to communities of interest? The results from this project have been disseminated to communities through publications in peer-reviewed journals, posters presented at scientific conferences and invited talks at local high schools and at the university. The dissemination of information also included formal lectures to students enrolled in the PI's courses SPSS4210 Plant Physiology: How Plants Work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During this funding period, we continued our effort to identify mutants with phenotypes related to vascular development, wood formation and organization of xylem tissues in the vascular and interfascicular regions. Progress has been made toward accomplish the goals. Objective 1. Characterize mutants with phenotypes related to secondary cell wall development. A dominant mutant showing a Xylem Development Inhibition (XDI) phenotype was identified and named xdi-d. Plant growth was affected in both vegetative and reproductive stages in the mutant lines. Compared to the wild-type (WT), the 3-week-old heterozygous (Het) and homozygous (Hom) plants were remarkably smaller in size. The main stems of mature heterozygous plants were approximately 20% shorter, and homozygous plants were 75% shorter than the wild type. The development of flowers, silique, and the number of stems were not significantly different from the wild type. Stem cross-sections were prepared from 6-week-old mature plants and stained with toluidine blue that stains all cell types, or with phloroglucinol that stains lignified cells with secondary cell walls. Xylem development was defective in both xdi-d heterozygous and homozygous plants resulting in much fewer xylem cells, especially in the protoxylem region. Metaxylem and protoxylem cells can be easily distinguished by their relative position and the size of their vessel cells. The xdi-d mutant plants were defective in protoxylem differentiation. To better characterize the xdi-d mutants, we also investigated the vascular and interfascicular fiber cells using transmission electron microscopy (TEM). In wild type plants, secondary cell walls of vascular fiber cells were well developed, and no cell contents were observed in these cells. In contrast, many parenchyma cells were observed next to the vessel cells in the xdi-d heterozygous and homozygous plants, indicating that the development of vascular fiber cells was affected in xdi-d mutants. The development of interfascicular fiber cells showed no significant difference between xdi-d mutants and the wild type plants. Objective 2. Identification of genes involved in regulating secondary wall formation. The expression level of AtAUR2 increased about 12 times and 20 times in xdi-d heterozygous and homozygous plants, respectively. To determine which gene(s) is responsible for the xdi-d phenotype, we performed over-expression experiments using each of the three genes. Overexpression of AtAUR2 driven by the Cauliflower Mosaic Virus (CaMV) 35S promoter in the wild type plants resulted in defects in proto-xylem and meta-xylem development and ectopic lignification of phloem fibers, which were similar to those of xdi-d mutants. A dwarf phenotype was also observed in the AtAUR2 overexpressing transgenic plants. The overexpression of AtAUR2 was confirmed with real time RT-PCR experiments. Therefore, these results indicated that the AtAUR2 gene was accountable for the phenotypes of xdi-d mutants. Objective 3. Characterization of the identified genes with biochemical and transgenic approaches. The expression of AtAUR1 and AtAUR2 was investigated with Promoter::GUS (β-glucuronidase) reporters. GUS staining results indicated that both Aurora genes were expressed in cotyledons and roots in seedling stage, with stronger staining in meristematic regions or fast growing tissues than mature tissues.Strong staining was observed in leaf veins, the vascular tissues in the leaves. In stem cross sections, GUS signals were observed in cambium region and protoxylem cells, as well as in cortex cells.To determine if the enhanced development of vascular tissues in aur1-2 aur2-2 plants are due to disruption of the AtAUR1 or AtAUR2 genes, we performed complementation experiments using AtAUR1 or AtAUR2 genomic sequences. The results indicated that the defects in plant growth can be fully rescued by either AtAUR1 or AtAUR2. The enhanced development in xylem and phloem regions were fully reverted to that of the wild type in plants expressing either AtAUR1 or AtAUR2. To understand if the enhanced xylem and phloem development of aur1-2aur2-2 plants is specific to vascular development, we performed complementation experiments using two constructs, ProSUC2:AUR2 and ProIRX3:AUR2, respectively. The promoters of SUC2 and IRX3 are known to be active in phloem and xylem, respectively. The transgenic studies indicated that the expression of AUR2 driven by SUC2 promoter (ProSUC2:AUR2) partially complemented plant growth phenotype of the aur1-2aur2-2 double mutant, and fully complemented the enhanced xylem phenotype. In contrast, expression of AUR2 under the IRX3 promoter could not complement the aur1-2aur2-2 double mutant phenotype. Constructs with the various mutated AtAUR2 genes were transformed into aur1-2 aur2-2 double mutants, respectively. The resulting transgenic plants were examined in F3 generation to determine the importance of these mutations to AtAUR2 function. For each construct, multiple representative lines were selected from approximately 30 independent transgenic lines. Three AtAUR2 mutations, i.e. G90/N, D166/A, and T179/A, failed to rescue the aur1-2 aur2-2 mutants. In comparison to the wild type, the transgenic plants developed very short stems, and the staining of transverse cross section showing enhanced secondary wall development and thicker phloem region. Expression analysis using real-time PCR indicated that the transgene expressed at similar or higher level than those of the wild-type (Fig. 9C). The G90 (glycine) is a critical amino acid for substrate binding, while D166 (aspartic acid) and T179 (threonine) are responsible for phosphorylation and auto-phosphorylation. These results clearly showed that the kinase activity including auto-phosphorylation, and substrate binding of the AtAUR2 protein are essential steps to its function in vascular development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Kwang-Hee Lee, Utku Avci, Liying Qi and Huanzhong Wang (2018). The Aurora kinases function in vascular development in Arabidopsis. Plant and Cell Physiology.DOI: https://doi.org/10.1093/pcp/pcy195
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Qian Du, Kwang-Hee Lee, Liying Qi and Huanzhong Wang (2018) Annual Plant Biology Symposium at UMass, The role of auxin in repressing secondary wall development in fibers of Arabidopsis Oct 13, 2018
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Huanzhong Wang, Kwang-Hee Lee, Qian Du, Liying Qi (2018) Plant Biology 2018 title The role of auxin in repressing secondary wall development in fibers of Arabidopsis Montreal, Quebec, July 14-18, 2018
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Kwang-Hee Lee, Utku Avci, Liying Qi and Huanzhong Wang (2018). Title The alpha Aurora kinases function in vascular development in Arabidopsis. 2018 Northeast ASPB Section annual meeting, April 28 - 29, 2018
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience: Target audiences include students at the Woodstock Academy, and students enrolled in the PI's courses, PLSC 4210 Plant Physiology, How Plants work and PLSC3230 Biotechnology, Science, Application, Impact, Perception. In addition, those reached through the paper that resulted from this project were also our target audiences. The PI was invited to give one guest lecture at Woodstock Academy titled "Biofuels, biomass and plant vascular stem cells" on May 18th 2017; and two guest lectures for the Sustainability 360 class at UConn on Nov 16, 2016 and April 17th, 2017, respectively; Students attending the PI's lectures for the Biotechnology course and the guest lectures were provided with information about biofuel production and environment impact of fossil fuels. Readers benefited from a paper published during the last funding period titled "Shoot Apical Meristem Size Measurement" in Bio-protocol. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities were given to graduate students and undergraduate students. One graduate student, Qian Du, worked on deciphering the functions of miR165B and has been investigating an additional gene that has a function in controlling vascular bundle numbers. She was trained in experimental design and various molecular techniques. Four undergraduate students participated in the project by helping care for the plants, collecting materials, and performing experiments. How have the results been disseminated to communities of interest?The results from this project have been disseminated to communities through one publication in peer-reviewed journals and invited talks in local high schools and in the university. Dissemination included formal lectures to students who enrolled in the PI's courses PLSC3230 Biotechnology, Science, Application, Impact, Perception and PLSC4210 Plant Physiology: How plants work. What do you plan to do during the next reporting period to accomplish the goals?We will continue cloning new genes in secondary cell wall development, and investigating their biological functions through biochemical and genetics studies. We will publish our results in peer-review journals, outreach to the scientific communities and the public with presentations at professional meetings.
Impacts
What was accomplished under these goals?
The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. in the past funding period, we continued our effort to identify new mutants showing phenotypes related to wood formation or organization of xylem tissues in the vascular and interfascicular regions. Progress has been made toward accomplish the goals. Objective 1. Characterize mutants with phenotypes related to secondary cell wall development. We identified additional mutant lines that showed altered secondary cell wall formation or disorganized vascular tissues. In Arabidopsis, Aurora kinases play important roles in primary meristem maintenance, but their functions in vascular development are still elusive. We identified a dominant xdi-d mutant showing xylem development inhibition. Gene cloning and transgenic over-expression experiments indicated that the activation of a AUR gene is responsible for the XDI phenotype. In contrast, the knock-out mutant plants showed enhanced differentiation of phloem and xylem cells, indicating the Aurora kinases negatively affect xylem differentiation. Key regulatory genes in vascular cell differentiation were upregulated in the knock-out mutants, but downregulated in xdi-d mutants, further support the functions of α Aurora kinases in vascular development. Gene mutagenesis and transgenic studies showed that protein phosphorylation and substrate binding, but not protein dimerization and ubiquitination, are critical for the biological function of AUR gene. These results indicate that α Aurora kinases play key roles in vascular cell differentiation in Arabidopsis. We will further investigate the function of this gene. Objective 2. Identification of genes involved in regulating secondary wall formation. A high-through-put transcriptome analysis on secondary cell wall development was performed along stem maturation in M. truncatula. Differently expression transcription factor genes were identified that may have important functions in xylem development in Medicago. These data has been published in BMC genomics. Objective 3. Characterization of the identified genes with biochemical and transgenic approaches. Several genes function in secondary cell wall development, such as XVP and AUR, are currently under study. We have analyzed their functions in detail using biochemical and transgenic approaches. The biological function of these genes is currently under investigation using transgenic studies. We expect that these analyses will further clarify the functions of the candidate genes in secondary cell wall biosynthesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Shoot Apical Meristem Size Measurement
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience: Target audiences include attendees at the PI's seminars at the Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Science (CAAS), the Plant Biology Discussion group at UConn, students at the Woodstock Academy, and participants in the Plant biology graduate program; and students who enrolled in the PI's courses PLSC3230 Biotechnology, Science, Application, Impact, Perception and PLSC3210/5210 Molecular Laboratory Techniques. Readers of the papers that resulted from this project and listed later in this report were also our target audiences. The PI gave talks for the aforementioned meetings and conferences. These include invited talks at the Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Science (CAAS) titled "Boosting Biofuels by switching on-off secondary cell wall development" on July 21, 2016;at the Plant Biology Discussion group at UConn "Biofuels, biomass and plant vascular stem cells" on May 19, 2016; at the Woodstock Academy "Biofuels, biomass and plant vascular stem cells" on Mar 18, 2016; at the Plant biology graduate program seminars at University of Massachusetts at Amherst titled "Boosting biofuels by switching on-off secondary cell wall development." on Dec 3, 2015. The students for the Biotechnology course were provided with information involved with biofuel production and environment impact of fossil fuels. The PI also applied materials created in this project in his teaching lab PLSC5210/3210. Students practiced molecular techniques using these materials. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunites were given to graduate students working on this project, and specifically on deciphering the functions of miR165B and other related genes. In addition, students were trained in various molecular techniques and experimental design. A master student also worked on the xvp project and has published one review paper regarding vascular development. In the meantime, four undergraduate students participated in the project by helping care for plants, collecting materials, and performing experiments. How have the results been disseminated to communities of interest?The results from this project have been disseminated to communities through three articles published in peer-reviewed journals and invited talks in regional, national and international meetings and conferences. In addition, formal lectures to students who enrolled in the PI's courses PLSC3230 Biotechnology, Science, Application, Impact, Perception and PLSC3210/5210 Molecular Laboratory Techniques included information on this research project. What do you plan to do during the next reporting period to accomplish the goals?We will continue our effort in cloning new genes in secondary cell wall development, and investigating biological functions of the cloned genes through biochemical and genetics studies. We will publish our results in peer-review journals and reach out to the scientific communities and the public with presentations.
Impacts
What was accomplished under these goals?
Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. Recent studies indicated that Class III homeodomain leucine zipper transcription factors (HD-ZIP III TFs) and microRNA 165/166 (miR165/166) may play important roles in SCW formation. Progress has been made toward accomplish the goals. 1) Characterizemutants with phenotypes related to secondary cell wall development. We reported the function of miR165b and discussed possible functions of class III HD-ZIP transcription factors in secondary cell wall development. In both vascular and interfascicular regions, vascular cambium are formed in the periphery of the existing secondary wall bearing vessel and fiber cells. Indeed, the expanded protoxylem and metaxylem in men1 was believed to be the results of promoting the activity of fascicular cambium and interfascicular cambium. Recently, we reported a miR165b activation-tagging line, stp-2d, which showed a dominant secondary cell wall thickening phenotype. The stems of stp-2d are much thinner than the wild type indicating that the cambium activity is unlikely enhanced in the stp-2d mutant. Actually, the activation tagging of miR165b in the stp-2d mutant resulted in secondary cell wall biosynthesis in pith cells, which are located in the center of the stem. Transgenic overexpression of a microRNA resistant AtHB15 (mAtHB15) further indicate that miR165b functions through AtHB15 in regulating secondary wall development in pith. In addition to functions of microRNA in secondary cell wall development, we made progress in elucidating the function of xvp gene. Objective 2. Identification of genes involved in regulating secondary wall formation. A systematic microarray assay and high through-put real time PCR analysis of secondary cell wall development were performed along stem maturation in Medicago truncatula. More than 11,000 genes were differentially expressed during stem maturation, and were categorized into 10 expression clusters. Among these, 279 transcription factor genes were correlated with lignin/cellulose biosynthesis, therefore representing putative regulators of secondary wall development. The b-ZIP, NAC, WRKY, C2H2 zinc finger (ZF), homeobox, and HSF gene families were over-represented. Gene co-expression network analysis was employed to identify transcription factors that may regulate the biosynthesis of lignin, cellulose and hemicellulose. As a complementary approach to microarray, real-time PCR analysis was used to characterize the expression of 1,045 transcription factors in the stem samples, and 64 of these were upregulated more than 5-fold during stem maturation. Reverse genetics characterization of a cellulose synthase gene in cluster 10 confirmed its function in xylem development. Objective 3. Characterization of the identified genes with biochemical and transgenic approaches. We have cloned the genes, such as miR165, XVP, and a protein kinase gene AUR2, which are responsible for the corresponding mutant phenotypes. We have been working on analyzing their functions in detail by biochemical and transgenic studies. Biological function of the identified genes will be investigated by transgenic studies. We expect that these assays will clarify the function of the candidate genes in secondary cell wall biosynthesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jung Hyun Yang and Huanzhong Wang* (2016) Molecular mechanisms for vascular development and secondary cell wall formation. Frontiers in Plant Science 7:356,
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Huanzhong Wang*, Jung Hyun Yang, Fang Chen, Ivone Torres-Jerez, Yuhong Tang, Mingyi Wang, Qian Du, Xiaofei Cheng, Jiangqi Wen, and Richard A. Dixon (2016). Transcriptome analysis of secondary cell wall development in Medicago truncatula. BMC Genomics 17:23
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Qian Du and Huanzhong Wang* (2015). The role of HD-ZIP III transcription factors and miR165/166 in vascular development and secondary cell wall formation. Plant signaling and Behavior 10:10
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Huanzhong Wang and Qian Du (2016) Molecular Control of Secondary Cell Wall Development. The Fourth International Conference on Plant Vascular Biology. Shenzhen China, July 19-23
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Qian Du and Huanzhong Wang (2016) Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis. ASPB Annual Meeting. Austin Texas, July 9-13
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience: During thisreporting period, the PI had the opportunity to engage a broad audience and disseminate the knowledge generated from this research through academic courses,invited talks, guest lectures and journal publications. The target audience for this reporting period included students who enrolled in the PI's courses PLSC3230 Biotechnology, Science, Application, Impact, Perception and HORT4650: Plant Tissue Culture; faculty, researchers and students who participated in the Plant Biology symposium 2015 at the University of Maryland; and readers interested in the topics of the papers published on this research. Students in the Biotechnology course were exposed to information involved with biofuel production and environmental impact of fossil fuels. The audience at thePlant Biology symposium at University of Maryland, included faculty, students and symposium participants, were given a talk by the PItitled, "Boosting biofuel by molecular regulation of Biomass formation". Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, theproject provided training opportunities for one master student and one PhD student working on the characterization of the stp-2d mutant. Several undergraduate students were also involved in this research by providing assistance taking careof the plants, collecting samples and running PCR etc. How have the results been disseminated to communities of interest?We have published two peer reviewed papers in high impact journals, and presented our results at twoprestigious Gordon Conference meetings. The PI also gave an invited talk at the Plant Biology Symposium 2015 at the University of Maryland. In addition, the research related materials were presented to students who were enrolled in the twocourses taught by the PI. What do you plan to do during the next reporting period to accomplish the goals?The PI and his research group will continue characterizing the mutants identified from the screening.We expect to elucidate the functions of XVP gene and othergenes have been cloned in this project. We will use multidisciplinary approaches to understand how these genescontrol the phenotypes related to vascular differentiation and secondary cell wall development.We expect that characterization of these mutants and genes will helpimprove the current biomass feedstocks for biofuel production.
Impacts
What was accomplished under these goals?
The main goal of this project is toidentify new genes and to characterize their functions in the regulation of wood formation in Arabidopsis. During this reporting period, we finished the characterization of one of the mutants, stp-2d. The accomplishment of this researchwere the publication of two peer reviewed papers in high impact journalsand two presentation (abstracts)attwo prestigious Gordon Conference series. We believe the scientific findings made significantcontribution to our understanding of how plants produce wood tissues, which is very important toassist the development of new strategies of improvingpoplar and other forestry woody plants for bioenergy applications. Objective 1. Characterization of mutants with phenotypes related to secondary cell wall development. In the last funding period, we continued the effort of identifying novel regulators of vascular development. We have have characterized over 13,000 mutant plants from an activation tagged population using stem cross section and UV microscopy. A few new mutants were identified from the screening. But our main focus in this period wasto characterize the stp-2d mutant.The dominant mutant stp-2d showedsecondary wall thickening inpith cells (STP). Immunohistochemistry assays confirmed accumulation of secondary cell walls in the pithcells of the stp-2d mutant. Activation of microRNA 165b (miR165b) expression is responsible for the STPphenotype, as demonstrated by transgenic over-expression experiments. The expression of three class IIIHD-ZIP transcription factor genes, including AtHB15, was repressed in the stp-2d mutant. Transgenic overexpressionof a mutant form of AtHB15 that is resistant to miR165-mediated cleavage reversed the stp-2dmutant phenotype to wild-type, indicating that AtHB15 represses secondary wall development in pith. Characterizationof two athb15 mutant alleles further confirmed that functional AtHB15 is necessary for retainingprimary walls in parenchyma pith cells. Expression analyses of cell-wall synthetic genes and wall-relatedtranscription factors indicated that a transcriptional pathway is involved in AtHB15 function. These resultsprovide new insight into the molecular mechanism of secondary cell-wall development. We were also continuing our analysis of thexvp-d mutant, which showedprecocious xylem differentiation and altered vascular patterning. The polarized periclinal division of the cambium cells and subsequent differentiation of the progeny cells form well-separated and wellorganized vascular tissues. In the xvp-d mutant plants, xylem and phloem cells are intercalated, and the division orientation of the cambium cell is disturbed. The mutant plants showed a severely disorganized vascular phenotype in hypocotyls. Furthermore, the division of cambium cells in the mutant plants was no longer in the periclinal orientation as that of the wild type. In addition to the defects of vascular development, the xvp-d mutants also displayed abnormality in overall plant morphology.The pleiotropic growth phenotypes of the xvp-d mutants suggested that the abnormal vascular patterning and precocious xylem differentiation affected normal function of the vascular tissues in water and nutrient transportation.We have cloned the XVP gene, which encodes a NAC domain transcriptoin factor. Transcription factors control the expression of their downstream genes. We are currently focusing on funcitonal analysis of this gene. Objective 2. Identification of genes involved in regulating secondary wall formation. Secondary wall development is correlated with the stem maturation process.To characterize the transcriptome profile during Medicagostem maturation, we collected stem samples for RNA extraction and subsequent microarray expressionanalyses. Five internodes, i.e. the aforementioned internodes2, 3, 5, 7, and 9 from the Medicago primary stem,were collected in three biological replicates.Analysis of the microarray results indicated that11,380 genes were significantly differentially expressed(p < 8.16 × 10-7 and fold change≥ 2) in the relativelymore mature internodes.To identify which of these genes are importantfor secondary cell wall development, we performedhierarchical clustering analysis. The differentially expressedgenes were placed into 10 clusters based on their expressionpatterns in the five investigated internodes.Cellulose and lignin are two of the major components ofthe secondary cell wall. These genes are primarily placed in cluster 10.In cluster 10alone, there are 279 TFs that are correlated with secondarycell wall development.There are 8 and 4 TFs in cluster 5and cluster 8, respectively. Among these 291 transcriptionfactors, the b-ZIP, NAC, WRKY, C2H2 ZF, homeobox, andHSF gene families were over-represented as determined byhypergeometric probability analysis. We also found that one of the probe setsin cluster 10, Mtr.5123.1.S1_at, has highest identity withAtIRX1/AtCesA8. We named the corresponding Medicagohomolog gene MtIRX1. To investigate the biological function of MtIRX1, we performed a reverse genetic analysis. We expect the reverse genetic characterization of MTIRX1 will provideproof of concept that the transcriptome data mayserve as a valuable resource for further secondary cell wallgene discovery in M. truncatula. Objective 3. Characterization of the identified genes with biochemical and transgenic approaches. To understand the biological function of the identified genes, we performed biochemical and transgenic analysis. Examination ofcross-sections from the stem base of 6-week-old plantsshowed that all pith cells were parenchyma cells in wildtypeplants, but the pith cell walls were lignifiedin the heterozygous and homozygous stp plants.To determine whether the ectopically lignifiedpith cells of stp-2d mutants also accumulated celluloseand xylan (the main hemicellulose in Arabidopsis), wecharacterized the stem cross-sections using histochemicalapproaches. To characterize cellulose accumulation, weperformed Calcofluor-white staining and histochemistryusing a carbohydrate-binding module (CBM3a) that bindscrystalline cellulose.The accumulationof xylan in the mutant pith cells was demonstratedby an immunohistochemical approach using a xylan-specificmonoclonal antibody CCRC-M149.To determine whether expression of miR165b wasincreased because of activation tagging, we performed aNorthern blot analysis. RNA samples prepared from seedlingsand stem tissues were used for this experiment.We found that mature miR165b was significantly up-regulatedin heterozygous and homozygous mutant stp-2d plants. To further investigate whether activation of miR165b isresponsible for the STP phenotype in the stp-2d mutant,we performed transgenic analyses. We over-expressedmiR165b in the wild-type background driven by a CaMV35S promoter.Weexamined stemcrosssectionsfrom transgenic lines using phloroglucinol staining.The pith cells were significantly more lignified comparedto the wild-type, suggesting ectopic secondarythickening of pith walls in these lines.Further analysis highlights an uncharacterized function of miR165 in regulating secondary wall development throughthe class III HD-ZIP transcription factor AtHB15.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Qian Du, Utku Avci, Shengben Li, Lina Gallego-Giraldo, Sivakumar Pattathil, Liying Qi, Michael G. Hahn and Huanzhong Wang (2015). Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis. Plant Journal, 83(3): 388400
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Qian Du and Huanzhong Wang (2015). The role of HD-ZIP III transcription factors and miR165/166 in vascular development and secondary cell wall formation Plant signaling and Behavior 10:10
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Qian Du, Utku Avci, Lina Gallego-Giraldo, Sivakumar Pattathil, Liying Qi, Michael G. Hahn and Huanzhong Wang (2015) Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis, Gordon Research Conference on Plant Metabolic Engineering held at Waterville Valley in Waterville Valley NH, July 19-24
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Qian Du, Utku Avci, Lina Gallego-Giraldo, Sivakumar Pattathil, Liying Qi, Michael G. Hahn and Huanzhong Wang (2015) Mechanisms of switching on/off secondary cell wall development, Gordon Research Conference on Plant Cell Walls held at Bentley University in Waltham MA, July 12-17
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audiences include students who enrolled in the PI's courses PLSC3230 Biotechnology, Science, Application, Impact, Perception and PLSC3210/5210 Molecular Laboratory Techniques; faculty, researcher and students in the Department of Animal Science; and participant of the Pan America Plants and BioEnergy 2014 Congress. The students for the Biotechnology courseare provided with information involved with biofuel production and environment impact of fossil fuels. The PI also applied materials created/used by this project in his teaching lab PLSC3210. Students practiced molecular techniques using these materials. Further, the PI gave an invited talks on "Transcriptional Regulation of cell wall and seed development" that related to this project for the faculty members and students of the Department of Animal Science. On June 5th, the PI gave another invited talk, titled "Identification of new regulators of cell wall development in Arabidopsis" for the attendees of the Pan America Plants and BioEnergy 2014 Congress. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training opportunities for two Graduate students. One master student, Jung Yang worked on the characterization of mutant xvp-d. The other student,Qian Du,a PhD student, wasresponsible for thecharacterization of the stp-2d mutant. How have the results been disseminated to communities of interest? The work on screening of cell wall developement related to mutant xvp-d was reported viaa guest lecturer. Invited talk on Pan America Plants and BioEnergy 2014 Congress titled "Identification of new regulators of cell wall development in Arabidopsis." Session B2. Genetic Improvement of Plant Biomass II, Guelph, Canada June 4 -7, 2014 Invited seminar titled "Transcriptional Regulation of cell wall and seed development" was given to the faculty and students in the Department of Animal Science on April 4, 2014. What do you plan to do during the next reporting period to accomplish the goals? We expect to elucidate the functions of genes that control the phenotypes of the identified mutant lines. The analyses include genetics, chemical and transgenic studies.We expect characterization of these mutants will provide insights into the regulation of biomass accumulation and help to improve biomass feedstocks.
Impacts
What was accomplished under these goals?
In this project, weused genetic approaches to identify genes that function in the regulation of wood formation in Arabidopsis. These resultscontribute to our understanding of how plants produce wood tissues and assistin thedevelopment of strategies to improve poplar and other forestry woody plants for bioenergy applications. Objective 1. Characterization of mutants with phenotypes related to secondary cell wall development. To identify novel regulators of vascular development, we have characterized over 10,419 mutant plants from an activation-tagged population using stem cross section and UV microscopy. We have identified two mutants with clear secondary cell wall development related phenotypes. The first mutant was dominant and showed precocious xylem differentiation and altered vascular patterning, there we named the mutant xvp-d. In the wild type plants, cambium cells are located between phloem (phl) and xylem cells (xyl). The polarized periclinal division of the cambium cells and subsequent differentiation of the progeny cells form well-separated and well-organized vascular tissues. In the xvp-d mutant plants, xylem and phloem cells are intercalated, and the division orientation of the cambium cell is disturbed. We can detect prematurely differentiated xylem cells as lignified small cells shown in the xvp-d stems. Although the heterozygous plants (Het) already showed the characteristic mutant abnormalities, homozygous plants (Hom) displayed much stronger phenotypes. The mutant plants had much fewer cells per vascular bundle compared to wild type plants, indicating that the mutants may be defective in stem cell proliferation. Further analysis indicated that the number of cells in each of the three tissue types, phloem (Phl), cambium (Cam) and xylem (Xyl), were significantly reduced. The Arabidopsis hypocotyl has an anatomic organization that is comparable to the stem of tree species, and undergoes significant secondary growth. To investigate the cambium function and vascular organization in the mutant hypocotyls, we performed cross section and chemical staining. The mutant plants showed a severely disorganized vascular phenotype in hypocotyls. We found that phloem tissues were interspersed with xylem cells in the heterozygous plants, and these disorganized tissues developed almost to the center of the hypocotyl in the homozygous plants. Furthermore, the division of cambium cells in the mutant plants was no longer in the periclinal orientation as that of the wild type. The xvp-d mutant plants have pleiotropic effects on plant growth. In addition to the defects of vascular development, the xvp-d mutants also displayed abnormality in overall plant morphology. In the vegetative growth stage, the mutant plants are significantly smaller compared to the control. Mutant leaves have shorter and narrower leaf lamina, and leaf edges are irregularly curved down. The mature mutant plants are dwarf compared to the wild type, with the xvp-d plants being one fourth of the height of the wild type. The main stem of the mutant plants are not as predominant as that of the wild type plants. Although the mutant plants seem bushy, the branch numbers are similar to the wild type. The flowers of xvp-d mutants are indistinguishable from those of the wild type, and siliques are about the same size as the wild type plants. The pleiotropic growth phenotypes of the xvp-d mutants suggested that the abnormal vascular patterning and precocious xylem differentiation affected normal function of the vascular tissues in water and nutrient transportation. The second mutant was also discovered through the screening effort. Mutant plants with Secondary wall Thickening in Pith cells (STP) accumulate more biomass per plant compared to wild type and provide research models for optimizing biomass feedstocks. A semi-dominant mutant showing STP phenotype was identified and was named as stp-2d. Pith cells of the heterozygous (stp-2d/+) mutant plants showed ectopic lignification starting from periphery pith cells close to the fascicular and interfascicular fibers toward the pith center. Characterization of cross sections form stem base of 6-week old plants showed that pith cells of the wild type plants were all parenchyma cells, while the pith cell walls are lignified in the heterozyogous plants. Pith cells of the homozygous stp-2d mutants also showed ectopic lignification. Phloroglucinol staining confirmed the UV microscopy results. The plant growth was affected in the mutant lines. The stem diameter of the heterozygous and homozygous are also smaller compared to the wild type. The 3-weeks old mutant plants, especially the homozygous mutant plants, are significantly smaller than the wild type. The mutant leaves are also narrower and shorter than the wild type. Mature plants of the mutant lines are dwarf compared to the wild type. To determine whether the lignified pith cells of stp-2d mutants also ectopically accumulate cellulose and xylan, we characterized the stem cross sections of wild type and mutant plants with histochemical approaches in collaborating with researcher from UGA. For checking the presence of cellulose, we used calcofluor white staining and immunohistochemistry using a carbohydrate-binding module CBM3a that binds crystalline cellulose. The mutant pith cells accumulate cellulose that indistinguishable from the fiber cells in the vascular bundles and interfascicular regions. We also detected xylan accumulation in the mutant pith cells by immunohistochemistry using monoclonal antibodies CCRC-M149. Detection of pectic polysaccharides using CCRC-M38, a monoclonal antibody binds to de-esterified homogalacturonan, indicated that the mutant pith cells are quite different from pith cells in wild type plants, which accumulate only primary cell wall similar to the epidermal and cortical cells. These results indicated that pith cells of the stp-2d mutants developed secondary cell walls similar to the fascicular and interfascicular fibers. Objective 2. Identification of genes involved in regulating secondary wall formation. Secondary wall development is correlated with the stem maturation process. We have analyzed the microarray data set obtained from earlier results focusing on transcription factor genes. We used co-expression analyses strategy and identifiedsome TFs, especially zinc finger, MYB, homeobox, b-ZIP and NAC domain family genes. The selected genes will be analyzed further to understand their biological function. Objective 3. Characterization of the identified genes with biochemical and transgenic approaches. We have cloned the genes responsible for mutant phenotypes and will analyze their function in detail by biochemical and transgenic studies. Biological function of the identified genes will be investigated by transgenic studies. We expect that these assays will clarify the function of the candidate genes in secondary cell wall biosynthesis.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Identification of new regulators of cell wall development in Arabidopsis,Huanzhong Wang, Qian Du, Liying Qi,Session B2. Genetic Improvement of Plant Biomass II, Guelph, Canada June 4 -7, 2014
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