Feeding different forms of methionine and rumen-protected methionine alters the incorporation of 15N into microbial protein in batch culture

Second Place in CFAES Undergraduate Research ForumCreating a balanced amino acid (AA) profile for rumen microbes increases efficiency of feed conversion into microbial protein , which decreases manure N. Methionine (MET) is a limiting AA in lactating dairy cows. A steady supply of MET reduces the need for rumen bacteria to synthesize AA from carbon skeletons. A previous study conducted in the OSU dairy lab evaluated the supplementation of 2-hydroxy-4-(methylthio) butanoic acid (HMB) isopropyl ester (HMBi; 0.11%), MET (0.097%), HMBi + MET (0.055% + 0.048%) and control (CON) to continuous culture (CC) fermenters. Extracellular MET pools were higher for HMBi treatments than MET, but microbial protein synthesis (MPS) did not differ. We hypothesized MET accumulation may be due to differences in bacterial utilization of MET stereoisomers, because isopropanol (ISO; hydrolyzed from HMBi) may affect membrane fluidity and HMBi is more slowly converted to MET. This expanded treatments (TRT) to CON (1), L-MET (2; 0.097%), D-MET (3; 0.097%), HMBi (4; 0.125%), HMB (5; 0.098%), 2×HMBi (6; 0.250%), ½HMBi + ½DL-MET (7; 0.063% + 0.049%), and HMB + ISO (8; 0.098% + 0.039%). We investigated the effects of the TRTs with batch culture (BC) fermentation. Protozoa were washed out of fermenters in the CC study but traditional BC inocula contain protozoa. Therefore, rumen fluid was collected from 2 Holstein cows and CC was used to prepare faunated (F) and defaunated (D) inocula for use in BC. In addition to TRTs, BC tubes were dosed with 15N enriched (NH4)2SO4 to compare incorporation of 15NH3 into MICROBIAL PROTEIN. Each TRT contained 6 replicates (D and F inocula each dosed to 3 replicates) with measurements taken at 0, 2, 8, and 24 h. Measurements included TCA soluble N (SN), 15NH3, NH3, peptides (PEP; SN–NH3), 15N enriched non-ammonia-N (NAN), and total NAN. SN, NH3, and PEP data were analyzed using the mixed procedure of SAS to determine main effects and interactions (other data in progress). The main effect of time was significant for SN, NH3, and PEP; inocula for SN (D=23.38, F=25.05 mg/dL) and NH3 (D=10.50, F=12.28 mg/dL); no other main effects were found. Interactions were found (data not shown) for inocula×TRT×time for NH3 (2h) and PEP (8, 24h), inocula×time for SN (2, 8h) and NH3 (2, 8h), and TRT×time for PEP (8, 24h). These data indicate possible differences in MET utilization but data completion will allow for more specific conclusions. Application of this data may improve feed efficiency of rumen microbes and dairy cows, which could reduce feed costs and N pollution.No embarg

Robert Milton Zollinger, M.D., teacher, surgeon, soldier, and farmer.

From Humble roots, Dr. Robert Milton Zollinger worked his way to a position in history among the giants of American surgery. He was born on September 4, 1903, in the central Ohio town of Millersport, the son of Elmira and William Zollinger. Neither of his parents had a high school education, but they supported education and always expressed a confidence that young Robert would be successful at anything he attempted.1 He had aspirations of attending West Point, a dream that was never fulfilled when he decided to be a surgeon. On being informed of his son’s intentions, Zollinger’s father bestowed on him advice that he ostensibly adhered to throughout his career: ‘‘If you are going to be a doctor, be a good one.’

Nuclear protein phosphatases with Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis

Perception of the plant steroid hormone brassinolide (BL) by the membrane-associated receptor kinase BRI1 triggers the dephosphorylation and accumulation in the nucleus of the transcriptional modulators BES1 and BZR1. We identified bsu1-1D as a dominant suppressor of bri1 in A abidopsis. BSU1 encodes a nuclear-localized serine-threonine protein phosphatase with an N-terminal Kelch-repeat domain, and is preferentially expressed in elongating cells. BSU1 is able to modulate the phosphorylation state of BES1, counter acting the action of the glycogen synthase kinase-3 BIN2, and leading to inc eased steady-state levels of dephosphorylated BES1. BSU1 belongs to a small gene family; loss-of-function analyses unravel the extent of functional overlap among members of the family and confirm the role of these phosphatases in the control of cell elongation by BL. Our data indicate that BES1 is subject to antagonistic phosphorylation and dephosphorylation reactions in the nucleus, which fine-tune the amplitude of the response to BL.Fil: Mora Garcia, Santiago. Salk Institute. Plant Biology Laboratory; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Howard Hughes Medical Institute; Estados UnidosFil: Vert, Gregory. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados UnidosFil: Yin, Yanhai. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados UnidosFil: Caño Delgado, Ana. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados UnidosFil: Cheong, Hyeonsook. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados UnidosFil: Chory, Joanne. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados Unido

IDN1 and IDN2 are required for de novo DNA methylation in Arabidopsis thaliana.

DNA methylation is an epigenetic mark affecting genes and transposons. Screening for mutants that fail to establish DNA methylation yielded two we termed "involved in de novo" (idn) 1 and 2. IDN1 encodes DMS3, an SMC-related protein, and IDN2 encodes a previously unknown double-stranded RNA-binding protein with homology to SGS3. IDN1 and IDN2 control de novo methylation and small interfering RNA (siRNA)-mediated maintenance methylation and are components of the RNA-directed DNA methylation pathway

BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are perceived by the plasma membrane-localized leucine-rich-repeat-receptor kinase BRI1. Based on sequence similarity, we have identified three members of the BRI1 family, named BRL1, BRL2 and BRL3. BRL1 and BRL3, but not BRL2, encode functional BR receptors that bind brassinolide, the most active BR, with high affinity. In agreement, only BRL1 and BRL3 can rescue bri1 mutants when expressed under the control of the BRI1 promoter. While BRI1 is ubiquitously expressed in growing cells, the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells. Loss-of-function of brl1 causes abnormal phloem:xylem differentiation ratios and enhances the vascular defects of a weak bri1 mutant. bri1 brl1 brl3 triple mutants enhance bri1 dwarfism and also exhibit abnormal vascular differentiation. Thus, Arabidopsis contains a small number of BR receptors that have specific functions in cell growth and vascular differentiation.Fil: Caño Delgado, Ana. Salk Institute. Plant Biology Laboratory; Estados Unidos. Howard Hughes Medical Institute; Estados UnidosFil: Yin, Yanhai. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados UnidosFil: Yu, Cong. University of Michigan; Estados UnidosFil: Vafeados, Dionne. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados UnidosFil: Mora Garcia, Santiago. Howard Hughes Medical Institute; Estados Unidos. Salk Institute. Plant Biology Laboratory; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cheng, Jin Chen. University of Michigan; Estados UnidosFil: Nam, Kyoung Hee. University of Michigan; Estados UnidosFil: Li, Jianming. University of Michigan; Estados UnidosFil: Chory, Joanne. Salk Institute. Plant Biology Laboratory; Estados Unidos. Howard Hughes Medical Institute; Estados Unido

ZINC-FINGER interactions mediate transcriptional regulation of hypocotyl growth in Arabidopsis

Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana. Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light–dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth

Co-targeting RNA Polymerases IV and V Promotes Efficient De Novo DNA Methylation in Arabidopsis.

The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation

Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor

Receptor tyrosine kinases control many critical processes in metazoans, but these enzymes appear to be absent in plants. Recently, two Arabidopsis receptor kinases-BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for brassinosteroids-were shown to autophosphorylate on tyrosines. However, the cellular roles for tyrosine phosphorylation in plants remain poorly understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is tyrosine phosphorylated in response to brassinosteroid perception. Phosphorylation occurs within a reiterated [KR][KR] membrane targeting motif, releasing BKI1 into the cytosol and enabling formation of an active signaling complex. Our work reveals that tyrosine phosphorylation is a conserved mechanism controlling protein localization in all higher organisms

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes