Source: SOUTH DAKOTA STATE UNIVERSITY submitted to
ROOT BIOLOGY AND RHIZOSPHERE INTERACTIONS INFLUENCING SOYBEAN PRODUCTIVITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005973
Grant No.
(N/A)
Project No.
SD00H543-15
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Feb 20, 2015
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Subramanian, SE.
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Plant Science
Non Technical Summary
Root lateral organs such as lateral roots (LRs) and symbiotic nodules are unique in that they arise from non-meristematic differentiated cells whereas lateral organs in the shoot arise from founder cells in the meristem. LRs are initiated in response to both developmental and primarily abiotic environmental cues (e.g. nutrients, mechanical stimuli). A better root architecture achieved through coordinated initiation of lateral roots promotes water and nutrient uptake efficiency. Leguminous plants initiate symbiotic nodule development in response to rhizobia-derived lipo chito-oligosaccharide nodulation (nod) signals specifically under nitrogen-deprived conditions. Biological nitrogen fixation in symbiotic nodules of crop legumes viz. soybean alleviates the use of environmentally damaging chemical fertilizers. Therefore, understanding the development of these root lateral organs in crop legumes viz. as soybean is crucial for enhanced management of agriculture, natural resources and the environment especially in South Dakota. We propose to identify transcripts and microRNAs (miRNAs) differentially expressed between lateral root primordia (LRP) and nodule primordia (NP) followed by functional analysis of selected candidates. It is our strong argument that our genome-enabled transcriptome, and silence-ome analysis would bridge the knowledge gap on distinct signaling mechanisms governing the development of LRs and determinate nodules.A number of microbes, specifically bacteria, colonize root surfaces and some of these contribute to plant protection, nutrient cycling, resource conservation and even protection against environmental stresses. Understanding the influence of rhizodeposits (compounds released by plant roots) on the composition of root surface microbial communities would help us elucidate plant-microbe communication in the rhizosphere. In turn, we might be able to formulate management practices (e.g. seed treatments) that positively impact plant productivity by influencing rhizosphere microbe composition. The goal of this proposal is to study the influence of isoflavonoids, a major group of compounds present in soybean rhizodeposits, on the composition of soybean root surface bacterial communities. We have developed methodology and resources to generate soybean plants with altered rhizodeposit isoflavonoid content and seek to analyze root surface bacterial communities in these plants by culture-dependent and independent methods.Advanced understanding of these processes is expected to help develop technologies to enhance sustainable production of soybean, a key food, feed and fuel crop in SD and the US.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011820105050%
2061820110050%
Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1100 - Bacteriology; 1050 - Developmental biology;
Goals / Objectives
Root lateral organs such as lateral roots (LRs) and symbiotic nodules are unique in that they arise from non-meristematic differentiated cells whereas lateral organs in the shoot arise from founder cells in the meristem. LRs are initiated in response to both developmental and primarily abiotic environmental cues (e.g. nutrients, mechanical stimuli). A better root architecture achieved through coordinated initiation of lateral roots promotes water and nutrient uptake efficiency. Leguminous plants initiate symbiotic nodule development in response to rhizobia-derived lipo chito-oligosaccharide nodulation (nod) signals specifically under nitrogen-deprived conditions. Biological nitrogen fixation in symbiotic nodules of crop legumes viz. soybean alleviates the use of environmentally damaging chemical fertilizers. Therefore, understanding the development of these root lateral organs in crop legumes viz. as soybean is crucial for enhanced management of agriculture, natural resources and the environment especially in South Dakota. We propose to identify transcripts and microRNAs (miRNAs) differentially expressed between lateral root primordia (LRP) and nodule primordia (NP) followed by functional analysis of selected candidates. It is our strong argument that our genome-enabled transcriptome, and silence-ome analysis would bridge the knowledge gap on distinct signaling mechanisms governing the development of LRs and determinate nodules.A number of microbes, specifically bacteria, colonize root surfaces and some of these contribute to plant protection, nutrient cycling, resource conservation and even protection against environmental stresses. Understanding the influence of rhizodeposits (compounds released by plant roots) on the composition of root surface microbial communities would help us elucidate plant-microbe communication in the rhizosphere. In turn, we might be able to formulate management practices (e.g. seed treatments) that positively impact plant productivity by influencing rhizosphere microbe composition. The goal of this proposal is to study the influence of isoflavonoids, a major group of compounds present in soybean rhizodeposits, on the composition of soybean root surface bacterial communities. We have developed methodology and resources to generate soybean plants with altered rhizodeposit isoflavonoid content and seek to analyze root surface bacterial communities in these plants by culture-dependent and independent methods.
Project Methods
Transcript profiling: We will isolate total RNA (including sRNAs) from primordia and control samples using Trizol reagent and generate oligo dT-primed cDNA from ~5 µg of total RNA. High throughput sequencing (HTS) for transcript profiling will be performed at the University of Missouri, Columbia. Briefly, cDNA molecules will be sheared (average fragment length ~400bp), adapters added at both ends, amplified and sequenced using Illumina (Solexa) single end sequencing (1 x 50nt read length) to obtain unbiased whole genome transcript profiling. With the availability of assembled soybean genome sequence, this read length is sufficient for reliable transcript profiling. The current instrumentation provides at least 200m passing reads per lane (using default base calling parameters of Illumina's pipeline). Therefore, we will multiplex (using different sequence tags) up to 6 libraries on a single lane to obtain ~30m reads per sample. We will sequence transcripts from all 4 tissue-types (3 replicates each) using 2 lanes of Illumina. The resulting reads information will be used to quantitatively analyze gene expression in different samples.Identification of primordium-specific/preferred transcripts: For gene expression analysis, we will map HTS reads to predicted mRNA sequences ("reference") obtained from soybean genome sequence (current version Glyma2.0; www.phytozome.net) using the tuxedo pipeline on the iPlant discovery environment. The utilities in this pipeline can directly import HTS data, perform multiplexed RNA-seq analysis (identifying replicates using sequence tags) and identify differentially expressed genes38. First, we will compare read counts of four established soybean house-keeping reference genes, Cons4, 6, 7 and 15, normalized to the total number of mapped reads in the respective library (e.g. reads per kb per million mapped; RPKM). Presumably, the normalized read counts of these genes will be comparable among the six different samples. If not, it might indicate difference in over all transcriptional activity in the different samples. In that case, we will use weighted trimmed mean of log expression ratios based normalization40 to compare and identify differentially expressed genes.Profiling miRNA expression: We will isolate sRNAs (15-30nt size) from ~20 µg total RNA, sequentially ligate adapters, reverse transcribe to cDNA and non-linearly amplify through a 15 cycle PCR. We have extensive expertise in generating small RNA libraries. HTS of sRNA libraries will be performed at the University of Missouri, Columbia. Similar to transcript profiling, we will multiplex samples on a single lane using different sequence tags to obtain ~10m reads per replicate. This would provide reliable quantitative information on miRNA expression. We will use a single lane of HiSeq to complete sequencing of small RNAs from all four tissue-sections (3 replicates each).Root surface preparations from transgenic composite plants: We will prepare hairy root composite plants for bacterial community studies by labeling transgenic roots (with a Tough-Tags™ label) and clipping off non-transgenic roots. We will plant them in SC soil and allow them to grow in a growth chamber (25?C, 250 µE/m2/s; 14h day). We will sample plants for root surface microbes at 7 and 21 days after planting. For sampling, we will remove plants from the soil and shake them well to remove loosely bound coarse soil clumps. Then, we will carefully separate transgenic roots (already labeled using Tough tags™) from the plant, sonicate (70W, 42Khz ultrasonic bath) each root individually in 50 ml tubes containing phosphate buffered saline (pH 7.2) with 0.025% Tween 20 (PBST) for 20 sec to remove and discard loosely adhered soil particles. We will then transfer the roots to a new tube of PBST and sonicate (70W, 42Khz) for 3 minutes to dislodge tightly adhered soil particles and microbial biofilms. We will then remove the roots and centrifuge the tubes (5000xg 10min) to collect microbes and soil. We will refer to this pellet as 'root surface preparation'.We will perform 3 biological replicates and for each replicate we will pool root surface preparations from multiple roots (~30 roots) to obtain a well-represented community of bacteria. We will examine preparations from Vector control, CHRi and IFSi transgenic roots in parallel to identify the composition of root surface bacterial communities using both culture and culture-independent methods. The robust nature of silencing we observed (e.g ~96% IFSi roots with no detectable isoflavones) would minimize variability between replicates. While the majority of soil bacteria are known to elude culturing by standard methods, our future experiments will rely heavily on specific cultures, necessitating our best isolation attempts.Culture-dependent method: We will plate serial dilutions of aliquots from root surface preparations on to three different media (1. R2A, 2. Soil extract medium and 3. VL55 with xylan) solidified with two different agents (agar and gellan) i.e. a total of 6 types of media. Based on previous experience, we believe that the above media should help obtain a comprehensive and representative group of isolates from root surface preparations. We expect to have a total of 2,160 plates (3 plant genotypes x 2 time-points x 6 media x 3 biological replicates x 4 dilutions x 5 plates) through the experiment. We will randomly select 4 colonies per plate of highest dilution showing > 20 colonies (2,160 colonies) for allocation to phylotype and future applications. We will culture colonies in 96 well plates in the respective media and use a high throughput method (e.g. QuickExtract bacterial DNA extraction kit, Epicenter biotechnologies) to isolate genomic DNA. We will then amplify 16S rRNA gene sequences (regions V1-V5 using primers 27Fand R903) and sequence amplicons using conventional Sanger sequencing.Culture-independent method: For culture-independent assay of root surface bacterial communities, we will isolate total DNA from root surface preparations using MOBIO soil DNA isolation kit. We will amplify V3-V5 region of 16S rRNA genes from these DNA preparations (using primers F357 and R907), perform a 15 cycle amplification to avoid quantitative bias and sequence amplicons using pyrosequencing (454 GS FLX+). Primers will be amended with 5' 454 adapter sequence and bar codes. This technology currently provides at least 500 bases read length and >650,000 high quality reads per run (www.454.com). We expect at least 35,000 reads per sample.Sequence libraries will be aligned using tools in the Pyrosequencing Pipeline at the Ribosomal Database Project (RDP; http://rdp.cme.msu.edu). Any sequence shorter than 400 bp, chimeras (detected using Pintail), or sequences containing more than 1 ambiguous base will removed, as will all sequences with homopolymers of >9 nt length. Output from alignment will be analyzed using a variety of tools in mothur. Outputs will include LIBSHUFF analysis to ascertain difference between samples, ACE and Chao 1 richness indicators, Shannon and Simpson diversity indicators, calculation of % shared OTU's and the Morisita Horn index. The Library Compare Tool from RDP will used to compare the libraries using the default settings. Comparison of bacterial community composition in roots with altered isoflavonoid profiles with that of control transgenic roots would enable us to identify a subset of microbes influenced specifically by subsets of isoflavonoid compounds. Comparison of bacterial community composition in roots with altered isoflavonoid profiles with that of control transgenic roots would enable us to identify a subset of microbes influenced specifically by subsets of isoflavonoid compounds.

Progress 02/20/15 to 09/30/19

Outputs
Target Audience:Scientific community including undergraduate and graduate students and post-doctoral associates, in particular in the fields of legume biology, plant-microbe interactions, and plant hormone biolog Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student (Paul Gaillard (Ph.D.) was trained in plant molecular biology, nodule development assays, data interpretation, quantitative imaging, image analysis, and development of associated methods and pipelines. How have the results been disseminated to communities of interest?The results were and are being disseminated through publications, and scientific presentations and discussions in scientific meetings. Next generation sequence data from the project was used in bioinformatics workshops (BioSNTR workshop, ~80 participants impacted) and in the classroom (PS735 Next Generation Sequence Analysis, ~12 students impacted; PS792 RNA-Seq Analysis, ~14 students impacted) to demonstrate the use of this technology for advanced discoveries in biology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots. (100% completed) We identified genes enriched in emerging nodules, mature nodules, emerging lateral roots, and young lateral roots in soybean by comparing global gene expression profiles between each of these organs and adjacent root segments. Hierarchical clustering and subsequent analysis of gene enrichment indicated that lateral root tissues were enriched in auxin responses and cell wall biosynthesis/modification pathways, while nodule tissues were enriched in phenylpropanoid, purine synthesis, and symbiosis pathways. Further analysis of hormone biosynthesis and signaling pathways identified distinct components of auxin biosynthesis and catabolism enriched in lateral roots vs. nodules. Specific gene family members encoding enzymes associated with auxin biosynthesis appeared to have diversified during evolution to have distinct organ-specific functions. Two specific gene families associated with reduction of free auxin levels, GH3 acyl conjugase and CYP83B1 cytochrome P450 monooxygenase, were selected for functional characterization. This was based on our previous discovery that overall auxin output might be reduced during nodule initiation in soybean. A CYP83B1 family member that acts in a pathway parallel to that of auxin biosynthesis, and therefore competes for substrates and regulates auxin level, was specifically enriched in nodule tissues leading to the hypothesis that this gene might reduce auxin biosynthesis during nodule development. We identified distinct nodule enrichment of three genes encoding auxin-deactivating GRETCHEN HAGEN 3 (GH3) indole- 3-acetic acid (IAA) amido transferase enzymes: GmGH3-11/12, GmGH3-14 and GmGH3-15. In vitro enzymatic assays showed that each of these GH3 proteins preferred IAA and aspartate as acyl and amino acid substrates, respectively. GmGH3-15 showed a broad substrate preference, especially with different forms of auxin. Suppression of GmCYP83B1 led to high auxin levels in nodules, a reduction in nodule numbers and size, and defective nodule vascular development suggesting that regulation of auxin homeostasis by GmCYP83B1 is crucial for proper nodule numbers and maturity. Silencing the expression of these GH3 genes in soybean composite plants led to altered nodule numbers, maturity, and size. Our results indicate that these GH3s are needed for proper nodule maturation in soybean. We discovered two crucial genes that regulated auxin biosynthesis and regulate nodule number and maturity. We also developed a robust computational framework to predict potential gene regulatory networks (GRNs) associated with root lateral organ development in soybean. The genome-scale expression dataset obtained from soybean root nodules and lateral roots was subjected to biclustering using QUBIC. Biclusters (BCs) and transcription factor (TF) genes with enriched expression in lateral root tissues were converged using different network inference algorithms to predict high confident regulatory modules that are repeatedly retrieved in different methods. The ranked combination of results from all different network inference algorithms into one ensemble solution identified 21 GRN modules of 182 co-regulated genes networks potentially involved in root lateral organ development stages in soybean. The pipeline correctly predicted previously known nodule- and LR-associated TFs, including the expected hierarchical relationships. The results revealed high scorer AP2, GRF5, and C3H co-regulated GRN modules during early nodule development; and GRAS, LBD41, and ARR18 co-regulated GRN modules late during nodule maturation. Our results identified 21 previously unknown regulators that likely play a crucial role in lateral organ development. Goal 2. Analysis of microRNA sequence data and degradome data from nodule tissues (100% completed). We constructed a small RNA library from soybean nodule and adjacent root tissues. Analysis of this dataset resulted in the identification of 14 families of previously unknown miRNAs and their corresponding targets. The results indicated that microRNA regulation might be a crucial step in determining the output from specific metabolic pathways and auxin-cytokinin signaling pathways during nodule development. Combined with knowledge on hormone biosynthesis and signaling pathways in Goal 1 and our prior results, our data indicated that a microRNA pathway regulates spatio-temporal balance between the plant hormones auxin and cytokinin to direct proper nodule development. We put together a construct that carried nuclear localized fluorescence sensors for auxin and cytokinin, and used two photon induced fluorescence microscopy for concurrent quantitative 3-dimensional imaging to determine cellular level auxin and cytokinin outputs and ratios in root and nodule tissues of soybean. Overlays of determined auxin/cytokinin ratios on specific root zones and cell types accurately reflected those predicted based on previously reported outputs for each hormone individually. Importantly, distinct auxin/cytokinin ratios corresponded to distinct nodule cell types, indicating a key role for these hormones in nodule cell type identity. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere, and Goal 4. The effect of hairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated?(100% completed) Rhizosphere soil fractions tightly associated with roots were isolated, and PCR amplicons from 16S rRNA gene variable regions V1-V3 and V3-V5 from these fractions were sequenced using 454. The resulting data was resolved using MOTHUR and vegan to identify bacterial taxa and evaluate changes in rhizosphere bacterial communities. The soybean rhizosphere was enriched in Proteobacteria and Bacteroidetes, and had relatively lower levels of Actinobacteria and Acidobacteria compared to bulk soil. Isoflavonoids had a small effect on bacterial community structure, and in particular on the abundance of Xanthomonads and Comamonads. The effect of hairy root transformation on rhizosphere bacterial communities was largely similar to un-transformed plant roots with approximately 74% of the bacterial families displaying similar colonization underscoring the suitability of this technique to evaluate the influence of plant roots on rhizosphere bacterial communities. However, hairy root transformation had notable influence on Sphingomonads and Acidobacteria. IMPACT: Our results identified two potential mechanisms by which auxin output is likely to be regulated during soybean nodule development, and revealed specific mechanisms by which they might act. In addition, we determined that the ratio between auxin and cytokinin, two key plant hormones, dictates the development of cell types with distinct function with in the nodule. We will use this knowledge to develop strategies to regulate auxin outputs in nodules to optimize nodule numbers and maturity to enhance nitrogen fixation. Examples include screening soybean varieties for variants in specific target genes to develop/generate soybean cultivars with optimized nitrogen fixation. Our results regarding soybean microbiome have helped catalog key bacterial taxa that are abundant in the soybean rhizosphere. This is useful for future studies aimed at evaluating the impact of specific management practices or agricultural chemicals on rhizosphere microbial activities. In addition, we have shown that hairy root plants are a suitable system to evaluate rhizosphere microbial diversity, although specific carefully designed controls are needed for proper interpretation of the results. The methods developed can be used to evaluate microbial community composition and activity to determine their contribution to soil health in different agricultural scenarios.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Adhikari S, Damodaran S, Subramanian S. 2019. Lateral root and nodule transcriptomes of soybean. Data 4 (2), 64.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Kafle A, Cope KR, Raths R, Yakha JK, Subramanian S, B�cking H, Garcia K. 2019. Harnessing soil microbes to improve plant phosphate efficiency in cropping systems. Agronomy. 9(3): 137.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Damodaran S, Dubois A, Xie J, Ma Q, Hindi� V, Subramanian S. 2019. GmZPR3d interacts with GmHD-ZIP III proteins and regulates soybean root and nodule vascular development. Int. J. Mol. Sci. 20, 827 (invited contribution).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sekaran U, McCoy C, Kumar S, Subramanian S. 2019. Soil microbial community structure and enzymatic activity responses to nitrogen management and landscape positions in switchgrass (Panicum virgatum L.). GCB Bioenergy. doi:10.1111/gcbb.12591
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: S Subramanian. 2019. Little RNAs go a long way: Long-distance signaling by microRNAs. Mol Plant 12: 18-20. (Invited Spot Light article).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: S Subramanian, S Pathak, S Damodaran, P Gaillard. 2019. Nodule zone-specific hormone balance in soybean. Noble Research Institute Seminar Series. Ardmore, OK. May, 5.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: S Subramanian, S Pathak, S Damodaran, P Gaillard. 2019. Spatio-temporal auxin-cytokinin balance during nodule development in soybean. University of Arizona Seminar Series. Tuscon, AZ. Feb 5.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: S Subramanian, P Gaillard, NPD Nurmalasari, S Smith. 2019. Spatio-temporal auxin-cytokinin balance during nodule development in soybean. Phenome 2019. Tuczon, AZ. Feb 4.


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

Outputs
Target Audience:Scientific community including undergraduate and graduate students and post-doctoral associates, in particular in the fields of legume biology, plant-microbe interactions, and plant hormone biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students (Paul Gaillard (Ph.D.) and Suresh Damodaran (Ph.D.) were trained in plant molecular biology, nodule development assays, data interpretation, and scientific communication. In addition Gaillard was trained in quantitative imaging, image analysis, and development of associated methods and pipelines. How have the results been disseminated to communities of interest?The results were and are being disseminated through publications, and scientific presentations and discussions in scientific meetings. Next generation sequence data from the project was used in bioinformatics workshops (BioSNTR workshop, ~80 participants impacted) and in the classroom (PS735 Next Generation Sequence Analysis, ~12 students impacted) to demonstrate the use of this technology for advanced discoveries in biology. What do you plan to do during the next reporting period to accomplish the goals?Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots; and Goal 2. Analysis of microRNA sequence data and degradome data from nodule tissues? Complete the analysis of microRNA and degradome datasets to identify miRNA-target pairs with distinct expression patterns in nodule tissues. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere, and Goal 4. The effect of hairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated Apply the developed methods to complete analysis of microbial taxa and gene expression patterns in agricultural soils or rhizospheres

Impacts
What was accomplished under these goals? Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots. (80% completed) Analysis of hormone biosynthesis and signaling pathways indicated that genes associated with auxin biosynthesis appeared to be active in both nodules and lateral roots of soybean except for a CYP83B1 ortholog that had nodule-specific enrichment. Based on observations in Arabidopsis and the expression pattern of this gene, we hypothesized that GmCYP83B1 might regulate auxin homeostasis acting in parallel to the indole pyruvate pathway. Indeed, suppression of GmCYP83B1 led to high auxin levels in nodules, a reduction in nodule numbers and size, and defective nodule vascular development suggesting that regulation of auxin homeostasis by GmCYP83B1 is crucial for proper nodule numbers and maturity. Inhibition of auxin biosynthesis in GmCYP83B1 silenced roots using yucasin rescued nodule numbers, but not maturity. We have discovered a key role for GmCYP83B1 in regulating auxin homeostasis during soybean nodule development. Goal 2. Analysis of microRNA sequence data and degradome data from nodule tissues (80% completed). Analysis of hormone biosynthesis and signaling pathways combined with prior results indicated that a microRNA pathway regulates spatio-temporal balance between the plant hormones auxin and cytokinin to direct proper nodule development. We put together a construct that carried nuclear localized fluorescence sensors for auxin and cytokinin, and used two photon induced fluorescence microscopy for concurrent quantitative 3-dimensional imaging to determine cellular level auxin and cytokinin outputs and ratios in root and nodule tissues of soybean. The use of nuclear localization signals on the markers, and nuclei segmentation during image processing, enabled accurate monitoring of outputs in 3D image volumes. The ratiometric method used here largely compensates for variations in individual outputs due to sample turbidity and scattering, an inherent issue when imaging thick root and nodule samples typical of many legumes. Overlays of determined auxin/cytokinin ratios on specific root zones and cell types accurately reflected those predicted based on previously reported outputs for each hormone individually. Importantly, distinct auxin/cytokinin ratios corresponded to distinct nodule cell types indicating a key role for these hormones in nodule cell type identity. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere, and Goal 4. The effect of hairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated?(80% completed) Rhizosphere soil fractions tightly associated with roots were isolated, and PCR amplicons from 16S rRNA gene variable regions V1-V3 and V3-V5 from these fractions were sequenced using 454. The resulting data was resolved using MOTHUR and vegan to identify bacterial taxa and evaluate changes in rhizosphere bacterial communities. The soybean rhizosphere was enriched in Proteobacteria and Bacteroidetes, and had relatively lower levels of Actinobacteria and Acidobacteria compared to bulk soil. Isoflavonoids had a small effect on bacterial community structure, and in particular on the abundance of Xanthomonads and Comamonads. The effect of hairy root transformation on rhizosphere bacterial communities was largely similar to untransformed plant roots with ~74% of the bacterial families displaying similar colonization underscoring the suitability of this technique to evaluate the influence of plant roots on rhizosphere bacterial communities. However, hairy root transformation had notable influence on Sphingomonads and Acidobacteria. IMPACT: Our results identified two potential mechanisms by which auxin output is likely to be regulated during soybean nodule development, and revealed specific mechanisms by which they might act. In addition, we determined that the ratio between auxin and cytokinin, two key plant hormones, dictates the development of cell types with distinct function with in the nodule. We will use this knowledge to develop strategies to regulate auxin outputs in nodules to optimize nodule numbers and maturity to enhance nitrogen fixation. Examples include screening soybean varieties for variants in specific target genes to develop/generate soybean cultivars with optimized nitrogen fixation. Our results on the microbiomes of soybean has helped catalog key bacterial taxa that are abundant in the soybean rhizosphere. This knowledge is useful for future studies aimed at evaluating the impact of specific management practices or agricultural chemicals on rhizosphere microbial activities. In addition, we have shown that hairy root plants are a suitable system to evaluate rhizosphere microbial diversity, although specific carefully designed controls are needed for proper interpretation of the results.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fisher J, Gaillard P, Fellbaum CR, Subramanian S, Smith S. (2018) Quantitative 3D Imaging of cell level auxin and cytokinin response ratios in soybean roots and nodules. Plant Cell Environ. 41(9):2080-2092.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Damodaran S, Westfall CS, Kisely BA, Jez JM, Subramanian S. (2017) Nodule-Enriched GRETCHEN HAGEN 3 Enzymes Have Distinct Substrate Specificities and Are Important for Proper Soybean Nodule Development. Int J Mol Sci. 18(12). (invited contribution).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Fisher J, Gaillard P, Fellbaum CR, Subramanian S, Smith S. (2018) In vivo quantitative imaging of auxin and cytokinin responses in soybean roots and nodules. MidWest ASPB meeting, March 3-4, Ames, IA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Fisher J, Gaillard P, Fellbaum CR, Subramanian S, Smith S. (2018) In vivo quantitative imaging of auxin and cytokinin responses in soybean roots and nodules. North American Symbiotic Nitrogen Fixation meeting, May 20-23, Winnipeg, Manitoba, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Fisher J, Gaillard P, Fellbaum CR, Subramanian S, Smith S. (2918) In vivo quantitative imaging of auxin and cytokinin responses in soybean roots and nodules. ASPB annual meeting, July 14-18, Montreal, Quebec, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S Subramanian, S Pathak, S Aryal, (2018) Cell type identity during nodule development. North American Symbiotic Nitrogen Fixation meeting, May 20-23, Winnipeg, Manitoba, Canada.


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

Outputs
Target Audience: Scientific community including undergraduate and graduate students and post-doctoral associates, in particular legume biology, plant-microbe interactions and plant hormone biology communities. Changes/Problems:Due to the high computational resources and time required for gene regulatory network prediction experiments, miRNA discovery experiments involving comparison ofmicroRNA, degradome, and global gene expresion profiles was delayed. What opportunities for training and professional development has the project provided?One graduate student (Mr. Suresh Damodaran)wastrained in plant molecular biology, gene knock-down methods, nodule development assays, interpretation and scientific communication. A graduate student (Ms. Laura White) was trained in plant-microbe interactions, generation of libraries to evaluate microbial community gene expression ("metatranscriptomics"), interpretation, and scientific communication. A post-doc (Dr. Shuchi Smita) was trained in bioinformatics methods associated with bi-clustering of large scale transcriptomic datasets, building of gene regulatory networks, identification of regulators of these GRNs, data interpretation, and scientific communication. How have the results been disseminated to communities of interest?The results were and are being disseminated through publications, and scientific presentations and discussions in scientific meetings. Next generation sequence data from the project was used in bioinformatics workshops and in the classroom to demonstrate the use of this technology for advanced discoveries in biology. What do you plan to do during the next reporting period to accomplish the goals?Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots; and Goal 2. Analysis of microRNA sequence data and degradome data from nodule tissues 1. Perform morphological, biochemical, and molecular characterization of nodule phenotypes in CYP83B1 and GH3 knock-down soybean composite plant roots. 2. Continued discovery of microRNAs and perform functional evaluation of their roles in soybean nodule development. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere (60% completed); and Goal 4. The effect of hairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated 1. Evaluate microbial composition of soils where differing sources of nitrogen were used for crop production. 2. Evaluate metatranscriptomes of soybean plants to determine potential effects of soybean root exudate compounds.

Impacts
What was accomplished under these goals? Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots; and Goal 2. Analysis of microRNA sequence data and degradome data from nodule tissues (65% completed) 1. The expression and enrichment of specific auxin biosynthesis, transport, and signaling components were probed in the organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots. Hierarchical clustering and subsequent analysis of gene enrichment indicated that lateral root tissues were enriched in auxin responses;and cell wall biosynthesis/modification pathways while nodule tissues were enriched in phenylpropanoid, purine synthesis and symbiosis pathways. Further analysis of hormone biosynthesis and signaling pathways identified distinct components of auxin biosynthesis and catabolism enriched in lateral roots vs. nodules. For example, specific gene family members encoding enzymes associated with auxin biosynthesis appeared to have diversified during evolution to have distinct organ-specific functions. Two specific gene families associated with reduction of free auxin levels, GH3 acyl conjugaseand CYP83B1 cytochrome P450 monooxygenase, were selected for functional characterization. This was based on our previous discovery that overall auxin output might be reduced during nodule initiation in soybean. 2. A CYP83B1 family member that acts in a pathway parallel to that of auxin biosynthesis and therefore competes for substrates and regulates auxin level was specifically enriched in nodule tissues leading to the hypothesis that this gene might reduce auxin biosynthesis during nodule development. Promoter:GUS assays suggested that this gene is specifically expressed in the nodule primordium and subsequently in the nodule parenchyma excluding the vascular bundles. This pattern was spatially complementary to that of auxin response gene expression underscoring the above hypothesis. Indeed, silencing the expression of this gene resulted in increased auxin-responsive gene expression and reduced number of nodules strongly supporting our hypothesis. 3. We identified distinct nodule enrichment of three genes encoding auxin-deactivating GRETCHEN HAGEN 3 (GH3) indole-3-acetic acid (IAA) amido transferase enzymes: GmGH3-11/12, GmGH3-14 and GmGH3-15. In vitro enzymatic assays showed that each of these GH3 proteins preferred IAA and aspartate as acyl and amino acid substrates, respectively. GmGH3-15 showed a broad substrate preference, especially with different forms of auxin. Promoter:GUS expression analysis indicated that GmGH3-14 acts primarily in the root epidermis and the nodule primordium where as GmGH3-15 might act in the vasculature. Silencing the expression of these GH3 genes in soybean composite plants led to altered nodule numbers, maturity, and size. Our results indicate that these GH3s are needed for proper nodule maturation in soybean. 4. Gene regulatory networks were constructed using the organ-specific transcriptome profiles dataset. Biclusters (Bus) and transcription factor (TFs) genes with specific expression in lateral root tissues were converged in different network inference algorithms to infer high confident regulatory modules that are repeatedly retrieved in different methods. The ranked combination of results from all different network inference algorithms into one ensemble solution identified 21 GRN modules of 182co-regulated genes networks potentially involved in lateral root organ development stages in soybean. Result revealed multiple high confident GRN modules that were regulated by AP2, GRF5 and C3H TFs during early nodule development; and GRAS, LBD41, and ARR18 TFs late during nodule maturation. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere (30% completed); and Goal 4. The effect ofhairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated (85% completed) 1. We had previously optimized methods for collection of rhizospere soil fractions from soybean roots. These were suitable for culturing of microbes or for isolation of community DNA for PCR. Additional methods were developed specifically for storage, and processing of soil samples for evaluation of microbial community gene expression. Three different preservation methods were evaluated. The storage of rhizosphere fractions directly in -80 deg C freezer was the most suited for subsequent isolation of high quality RNA, than the use of Trizol or RNA later preservatives. Similarly, three different RNA amplification methods were evaluated and the random priming and universal adapter based amplification method (SEQR kit from Sigma-Aldrich) was found to be the most suitable in amplification of RNA. The optimized methodswould help identify microbial pathways active in specific rhizosphere and field soil samples through metatranscriptome analysis. IMPACT: Our results identified two potential mechanisms by which auxin output is likely to regulated during soybean nodule development: (i) deactivation of auxin levels by acyl conjugase GH3s and (ii) regulation of auxin levels via tryptophan substrate competition by CYP83B1.The data has provided key marker genes that can be used to select soybean varieties with optimal numbers of nodules. In addition, the results suggest that auxinic herbicides are likely to affect nodule development in addition to foliar injury. This knowledge can be used in additional basic research as well as evaluation of key mangement practices to mitigate auxin herbicide injury.

Publications

  • Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: S. Subramanian (2017) Hairy Root Composite Plant Systems in Root- 2 Microbe Interaction Research. S. Malik (ed.), Production of Plant Derived Natural Compounds through Hairy Root Culture, Springer International Publishing. https://doi.org/10.1007/978-3-319-69769-7
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: White LJ, Ge X, Br�zel VS, Subramanian S. (2017) Root isoflavonoids and hairy root transformation influence key bacterial taxa in the soybean rhizosphere. Environ Microbiol. 19(4): 1391-1406. doi: 10.1111/1462-2920.13602.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: White LJ, Ge X, Br�zel VS, Subramanian S. Soybean Rhizosphere Bacterial Community Structure as Influenced by Root Isoflavonoids. NE EPSCoR: Genomics to Phenome meeting, April 6-7, 2017, Lincoln, NE (presented by Laura White)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Damodaran S, Subramanian S. GmCYP83B1-mediated auxin flux regulation is crucial for soybean nodule development, NE EPSCoR: Genomics to Phenome meeting, April 6-7, 2017, Lincoln, NE (presented by Suresh Damodaran)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: S Smita, J Kiehne, E Zeng, Q Ma, S Subramanian. microRNA and Gene Regulatory Networks Governing Soybean Nodule Development, Plant and Animal Genome Conference (Genomics workshop), Jan 13-18, 2017, San Diego, CA (presented by Sen Subramanian)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: S Damodaran, S Subramanian. Regulation of Local Auxin Metabolism during Soybean Nodule Development. Plant and Animal Genome Conference (Legumes workshop), Jan 13-18, 2017, San Diego, CA (presented by Sen Subramanian)


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientific community including undergraduate and graduate students andpost-doctoral associates, in particular legume biology, plant-microbe interactions and next-generation sequencing communities. Changes/Problems:Interpretation of results from gene regulatory network analyses performed in the current reporting period were not completed due to unexpected delays resulting from software crashes and the need for comparison of results from multiple recently developed approaches. What opportunities for training and professional development has the project provided? Two graduate students, Sajag Adhikari (Ph.D.) andSuresh Damodaran (Ph.D.) were trained in plant molecular biology, analysis of pathways in high throughput sequencing data (RNA-seq), interpretation and scientific communication (written and oral). A graduate student, Laura White (Ph.D)was trained in plant-microbe interactions, taxonomic analysis of high throughput sequencing data ("metagenomics"), bacterial phylogeny, interpretation, and scientific communication (written and oral). How have the results been disseminated to communities of interest? The results were and are being disseminated through publications, and scientific presentations and discussions in scientific meetings. Next generation sequence data from the project was used in bioinformatics workshops and in the classroom to demonstrate the use of this technology for advanced discoveries in biology. What do you plan to do during the next reporting period to accomplish the goals?Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots; andGoal 2. Analysis of microRNA sequence data and degradome data from nodule tissues (50% completed) We will perform functional analysisof keyhormonebiosynthesis and signaling identifiedfrom pathway analysis oftheglobal gene expression datasets.Wewill perform gene regulatory network construction and analysis usingthehigh throughput sequencing data acomparisonsbetween the microRNA,degradome,andgloblageneexpresion profilestodetermine key microRNA genes during nodule development. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere(30% completed); andGoal 4. The effect of hairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated (70% completed) We will evaluate the soybean rhizosphere microbiome through studies on bacterial gene expression using metatranscriptomics, and canges in microbiome taxa in response to key rhizodeposit compounds and management products.

Impacts
What was accomplished under these goals? Goal 1. Evaluation of hormone biosynthesis and signaling pathways in organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots (30% completed). The results indicated that distinct auxin-cytokinin profiles were active during the formation of nodules vs. lateral roots suggesting that they do not share common developmental mechanisms. This observation also indicated that nodules borrowed little if any developmental pathways from lateral roots during evolution. Goal 2. Analysis of microRNA sequence data and degradome data from nodule tissues (50% completed) This resulted in the identification of 14 families of previously unknown miRNAs and their corresponding targets. The results indicated that microRNA regulation might be a crucial step in determining the output from specific metabolic pathways and auxin-cytokinin signaling pathways during nodule development. Goal 3. Taxonomic analysis of 16S amplicons from soybean rhizosphere(30% completed) The results showed that the soybean rhizosphere was enriched in Proteobacteria and Bacteroidetes, and had relatively lower levels of Actinobacteria and Acidobacteria compared to bulk soil. Isoflavonoids had a small effect on bacterial community structure, and in particular on the abundance of Xanthomonads and Comamonads. Goal 4. The effect of hairy root transformation, a technique routinely used in legume biology, on rhizosphere bacterial communities was evaluated (70% completed) The microbiome profiles of hairy roots were largely similar to untransformed plant roots with ~74% of the bacterial families displaying similar colonization underscoring the suitability of this technique to generate transgenic material to evaluate the influence of plant roots on rhizosphere bacterial communities. However, hairy root transformation had notable influence on Sphingomonads and Acidobacteria.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Nizampatnam, N. R., Schreier, S. J., Damodaran, S., Adhikari, S. and Subramanian, S. (2015), microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development. Plant J, 84: 140153. doi:10.1111/tpj.12965
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: R. Chintala, S. Subramanian, A.-M. Fortuna and T.E. Schumacher, Chapter 6 - Examining Biochar Impacts on Soil Abiotic and Biotic Processes and Exploring the Potential for Pyrosequencing Analysis, In Biochar Application, Elsevier, 2016, Pages 133-162, ISBN 9780128034330, http://dx.doi.org/10.1016/B978-0-12-803433-0.00006-0. (http://www.sciencedirect.com/science/article/pii/B9780128034330000060)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: S Damodaran, S Subramanian (2016) Quantitative Amplification of Cleaved Ends (qACE) to Assay MiRNA-directed Target Cleavage, Plant & Animal Genome Conference, San Diego, CA. Jan 12, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: S Adhikari, S Damodaran, S Subramanian (2016) Comparative Transcriptomics Reveals Distinct Transcription Factors and Hormone Action Pathways in Nodules and Lateral Roots of Soybean. Plant & Animal Genome Conference, San Diego, CA. Jan 12, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: LJ White, VS Brozel, S Subramanian (2016) Soybean Rhizosphere Bacterial Community Structure as Influenced by Root Isoflavonoids. Soybean meeting, Columbus, OH. Aug 6, 2016


Progress 02/20/15 to 09/30/15

Outputs
Target Audience: Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student was trained in plant molecular biology, analysis of high throughput sequencing data (RNA-seq), interpretation and scientific communication. A graduate student was trained in plant-microbe interactions, method development for isolation of root surface microbes, analysis of high throughputsequencing data ("metagenomics"), bacterial phylogeny, interpretation and scientific communication. How have the results been disseminated to communities of interest?The results were and are being disseminated through publications, and scientific presentations and discussions in scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We will continue analysis of the global transcriptome profiles of nodules and lateral roots to identify specific hormonal signaling and biosynthesis pathways that are active in each organ. We will perform gene regulatory network analysis to identify key regulatory circuits active in these organs. We will identify bacterial species influenced by isoflavonoids in soybean roots and evaluate their potential roles in the soybean rhizosphere for increased plant productivity.

Impacts
What was accomplished under these goals? 1. We have identified organ-specific transcriptome profiles of emerging nodules, mature nodules, emerging lateral roots and young lateral roots by comparison to adjacent root tissues of appropriate age 2. We identified that nodules and lateral roots have distinct sets of expression even at the earliest stages of development that were compared. The most common biological process common between these organs was cell division and replication. 3. Our results indicate that several known signaling elements involved in the initiation of lateral roots were poorly expressed or not enriched in nodules 4. We also show that distinct sets of transcription factor families are enriched in LR vs. nodule tissues 5. Root surface preparations were obtained from soybean plans with reduced levels of isoflavonoids and PCR amplicons were sequenced using high throughput sequencing methods. 6. Results indicate that isoflavonoids influence a small group of bacterial phylotypes in the soybean rhizosphere

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: LJ White, K Jothibasu, RN Reese, VS Br�zel, S Subramanian (2015) Spatio temporal influence of isoflavonoids on bacterial diversity in the soybean rhizosphere. Molecular Plant-Microbe Interactions 28 (1), 22-29
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: LJ White, VS Brozel, S Subramanian (2015) Isolation of Rhizosphere Bacterial Communities from Soil. Bio-Protocol 5 (16), e1569
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: S Subramanian (2015) Did root nodules evolve from shoot lateral organs? Evidence from transcriptomics and miR160 action in soybean. 23rd North American Nitrogen Fixation Conference, Ixtapa, MX, Dec6-10 2015.