Divisions of labor in the thiamin biosynthetic pathway among organs of maize

The B vitamin thiamin is essential for central metabolism in all cellular organisms including plants. While plants synthesize thiamin de novo, organs vary widely in their capacities for thiamin synthesis. We use a transcriptomics approach to appraise the distribution of de novo synthesis and thiamin salvage pathways among organs of maize. We identify at least six developmental contexts in which metabolically active, non-photosynthetic organs exhibit low expression of one or both branches of the de novo thiamin biosynthetic pathway indicating a dependence on inter-cellular transport of thiamin and/or thiamin precursors. Neither the thiazole (THI4) nor pyrimidine (THIC) branches of the pathway are expressed in developing pollen implying a dependence on import of thiamin from surrounding floral and inflorescence organs. Consistent with that hypothesis, organs of the male inflorescence and flowers are shown to have high relative expression of the thiamin biosynthetic pathway and comparatively high thiamin contents. By contrast, divergent patterns of THIC and THI4 expression occur in the shoot apical meristem, embyro sac, embryo, endosperm, and root-tips suggesting that these sink organs acquire significant amounts of thiamin via salvage pathways. In the root and shoot meristems, expression of THIC in the absence of THI4 indicates a capacity for thiamin synthesis via salvage of thiazole, whereas the opposite pattern obtains in embryo and endosperm implying that seed storage organs are poised for pyrimidine salvage. Finally, stable isotope labeling experiments set an upper limit on the rate of de novo thiamin biosynthesis in maize leaf explants. Overall, the observed patterns of thiamin biosynthetic gene expression mirror the strategies for thiamin acquisition that have evolved in bacteria

A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses

In rice, the class I small heat shock protein (sHSP-CI) genes were found to be selectively induced by L-azetidine-2-carboxylic acid (AZC) on chromosome 3 but not chromosome 1. Here it is shown that a novel cis-responsive element contributed to the differential regulation. By serial deletion and computational analysis, a 9 bp putative AZC-responsive element (AZRE), GTCCTGGAC, located between nucleotides –186 and –178 relative to the transcription initiation site of Oshsp17.3 was revealed. Deletion of this putative AZRE from the promoter abolished its ability to be induced by AZC. Moreover, electrophoretic mobility shift assay (EMSA) revealed that the AZRE interacted specifically with nuclear proteins from AZC-treated rice seedlings. Two AZRE–protein complexes were detected by EMSA, one of which could be competed out by a canonical heat shock element (HSE). Deletion of the AZRE also affected the HS response. Furthermore, transient co-expression of the heat shock factor OsHsfA4b with the AZRE in the promoter of Oshsp17.3 was effective. The requirement for the putative AZRE for AZC and HS responses in transgenic Arabidopsis was also shown. Thus, AZRE represents an alternative form of heat HSE, and its interaction with canonical HSEs through heat shock factors may be required to respond to HS and AZC

Study of Class I Small Heat Shock Protein Gene Family in Rice (Oryza sativa Tainung No. 67): Characterization, Expression, and Regulation

第一族低分子量熱休克蛋白質（class I small heat shock protein, sHSP-CI）是植物於熱逆境下累積量及種類最多的熱休克蛋白質，本研究針對水稻(Oryza sativa Tainung No.67)中sHSP-CI基因群進行詳細的基因鑑定及基因表現分析。水稻sHSP-CI基因群成員總共有九個，分別為Oshsp16.9A、Oshsp16.9B、Oshsp16.9C、Oshsp16.9D及Oshsp17.9B位於第一號染色體上，而Oshsp17.3、Oshsp17.7、Oshsp17.9A及Oshsp18.0則位於第三號染色體上，這九個基因在編譯區(coding region)具有很高的相似度(>60 %)，而歧異度較高的區域則位於其3’-UTRs (<40%)，根據α-helix 2 (α2) 氨基酸序列的變異性，我們建議進一步將單子葉植物的sHSP-CI基因群分成subclass A及subclass B兩群。利用二維膠體電泳分析各個基因的in vitro transcription/translation產物或者重組蛋白質，已經確認這九個基因產物在二維膠體上pI值及分子量的相對應位置以及構成heat shock complex (HSC)的主要基因成員。以RT-PCR分析各個水稻class I sHSP基因在熱逆境下的表現，在熱處理下，除了Oshsp17.9B之外，其它基因皆會被誘導大量表現，位於第三號染色體上的sHSP-CI基因在32℃與41℃下很迅速地被誘導表現，而相同處理下第一號染色體上的sHSP-CI基因則較慢被誘導表現。以砷處理白化幼苗，除了Oshsp16.9D和Oshsp17.9B之外，其餘七個sHSP-CI基因皆會誘導表現，但是以第三號染色體上sHSP-CI基因的表現量較多。在鎘、azetidine-2- carboxylic acid（Aze, proline的類似物）及canavanine（Can, arginine的類似物）處理之下則只觀察到第三號染色體上的sHSP-CI基因被誘導表現，相同的sHSP-CI基因表現形式也可受到酒精、NaCl、H2O2和CuCl2引發。此外，我們也觀察到在水稻種子發育成熟過程中只有Oshsp16.9A大量表現，顯示此蛋白質在發育中扮演重要角色。藉由報導基因短暫性表現分析顯示Oshsp17.3和Oshsp18.0共享一個356 bp的雙向啟動子（bidirectional promoter），由此分析亦證明Aze所造成的選擇性差異表現確實是源自於水稻sHSP-CI基因啟動子的原本特性，亦即水稻sHSP-CI基因的選擇性差異表現最有可能是受轉錄層次的調節，進一步由Oshsp17.3啟動子剔除分析確認一段對Aze有專一性反應元素（cis-responsive element, GTCCAGGACG）位在相對於轉錄啟始點-181至-171間。此外也發現一水稻OsMAPK2基因mRNA會受Aze誘導而增加約3倍的表現量，而鎘則只增加約1.5倍。綜合目前的結果顯示已經可以知道氨基酸類似物會以不同於熱休克的方式誘導調節水稻sHSP-CIs基因轉錄表現。最後我根據本研究所得的結果與前人關於訊息傳導的研究，提出一個訊息傳導的可能模式來解釋熱訊息傳導與化學誘導物如Aze或鎘訊息傳導之間的互相關聯性。The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of 9 members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. The rice sHSP-CI genes share high homology in the coding regions (>60%) and low homology in the 3’-UTRs (<40%). According to the amino acid variation of theAbstract in Chinese I Abstract in English III Abbreviations V Introduction 1. Heat Shock (HS) Response and HSPs 2 2. The Biological Significance of sHSPs in Plants 3 3. Diversity and Classification of Plant sHSP Family 5 4. Physical-Chemical Properties and Structure of Plant sHSPs 9 5. Chaperone Mechanism of sHSP Action in Plants 12 6. Plant sHSP Expression during Heat Stress 16 7. Expression of Plant sHSP under Normal Growth Conditions 18 7.1 Constitutive Expressions in Vegetative Tissues 18 7.2 Constitutive Expressions in Reproductive Tissues 19 8. Expression of sHSPs in Response to Other Environmental Stresses 21 8.1 Osmotic Stress 21 8.2 Oxidative Stress 22 8.3 Chilling Stress 22 8.4 Chemical Inducers 23 8.5 Other Stresses 24 9. Structural Features Required for Regulation of Plant sHSP Genes 24 9.1 The Essential Cis-response Elements in Promoter Regions 25 9.2 Cis-responsive Motifs in the Enhancer Regions 25 9.3 The Function of 5'-Untranslated Regions of Plant sHSP Genes 26 10. Transcriptional Regulation of sHSP Genes by HSFs 27 11. Developmental Regulation of Plant sHSPs by HSFs 31 12. Signal Transduction Pathway of Plant sHSP Gene Expression 33 13. Progresses and Goals 37 13.1 Progresses of Research Project in Recent Ten Years 37 13.2 Goals of Current Research Project 38 Materials and Methods 1. Plant Materials 41 2. Chromosome Mapping 41 3. RNA Isolation and RT-PCR 41 4. Primer Extension Analysis 43 5. Coupled in Vitro Transcription/Translation and Expression of Recombinant Proteins 43 6. Two Dimensional Gel Electrophoresis (2-DE) and Western Blotting Analysis 44 7. Particle Bombardment and Transient Expression Assays 45 8. Preparations of DNA Constructs for Promoter Deletion Analysis 47 9. Bioinformatics 47 Results 1. Rice sHSP-CI Gene Family Contains Nine Members 48 2. Sequence Analysis of the Rice sHSP-CI Gene Family 48 3. Genomic Organization and Promoter Sequences of the Rice sHSP-CIs Gene Family 49 4. Seven Major sHSP-CI Are Present in the Rice HSCs 51 5. The Heat Stress Responsiveness of the Rice sHSP-CI Genes 52 6. Rice sHSP-CI Genes Are Induced by Various Chemical Inducers in a Selective Manner 53 7. ROS May Have a Role in the Selective Induction of Rice sHSP-CI Genes in Response to Chemical Inducers 54 8. Transient Expression Assays of the Promoter Activity Supported the in Vivo Selective Expression of Rice sHSP-CI Genes by Aze Treatment 54 9. Specific Members of Rice sHSP-CIs Are Induced During Seed Development 55 10. OsMAPK2 Transcripts Are Enhanced by Aze Treatment 55 11. Requirement of a Specific Cis-Responsive Element for Induction of Oshsp17.3 by Aze treatment 56 Discussions 1. Rice sHSP-CI Gene Family 58 2. Structural Features of Monocot sHSP-CIs 59 3. Genomic Organization of the Rice sHSP-CI Gene Family 60 4. Oshsp17.3 and Oshsp18.0 Share a Bidirectional Promoter 61 5. HS Response 63 6. HS-like Response Induced by Chemical Inducers 64 7. Oshsp16.9A Is Developmentally Regulated During Rice Grain Maturation 65 8. Role of H2O2 in the Selective Induction of Rice sHSP-CI Genes 66 9. OsMAPK2 May Be Involved in the Selective Induction of Rice sHSP-CI Genes 67 10. GTCCTGGACG motif (Box1) May Be an Aze-Responsive Element (AZRE) Involved in the Selective Induction of Rice sHSP-CIs by Aze 68 11. A Model of Oshsp17.3 and Oshsp18.0 Induction by Aze and Its Relationship with HS 70 Prospectus 72 Tables 74 Figures 77 References 9

Sugar modulation of anaerobic-response networks in maize root tips

Sugar supply is a key component of hypoxia tolerance and acclimation in plants. However, a striking gap remains in our understanding of mechanisms governing sugar impacts on low-oxygen responses. Here, we used a maize (Zea mays) root-tip system for precise control of sugar and oxygen levels. We compared responses to oxygen (21 and 0.2%) in the presence of abundant versus limited glucose supplies (2.0 and 0.2%). Low-oxygen reconfigured the transcriptome with glucose deprivation enhancing the speed and magnitude of gene induction for core anaerobic proteins (ANPs). Sugar supply also altered profiles of hypoxia-responsive genes carrying G4 motifs (sources of regulatory quadruplex structures), revealing a fast, sugar-independent class followed more slowly by feast-or-famine-regulated G4 genes. Metabolite analysis showed that endogenous sugar levels were maintained by exogenous glucose under aerobic conditions and demonstrated a prominent capacity for sucrose re-synthesis that was undetectable under hypoxia. Glucose abundance had distinctive impacts on co-expression networks associated with ANPs, altering network partners and aiding persistence of interacting networks under prolonged hypoxia. Among the ANP networks, two highly interconnected clusters of genes formed around Pyruvate decarboxylase 3 and Glyceraldehyde-3-phosphate dehydrogenase 4. Genes in these clusters shared a small set of cis-regulatory elements, two of which typified glucose induction. Collective results demonstrate specific, previously unrecognized roles of sugars in low-oxygen responses, extending from accelerated onset of initial adaptive phases by starvation stress to maintenance and modulation of co-expression relationships by carbohydrate availability

Sugar modulation of anaerobic-response networks in maize root tips

Sugar supply is a key component of hypoxia tolerance and acclimation in plants. However, a striking gap remains in our understanding of mechanisms governing sugar impacts on low-oxygen responses. Here, we used a maize (Zea mays) root-tip system for precise control of sugar and oxygen levels. We compared responses to oxygen (21 and 0.2%) in the presence of abundant versus limited glucose supplies (2.0 and 0.2%). Low-oxygen reconfigured the transcriptome with glucose deprivation enhancing the speed and magnitude of gene induction for core anaerobic proteins (ANPs). Sugar supply also altered profiles of hypoxia-responsive genes carrying G4 motifs (sources of regulatory quadruplex structures), revealing a fast, sugar-independent class followed more slowly by feast-or-famine-regulated G4 genes. Metabolite analysis showed that endogenous sugar levels were maintained by exogenous glucose under aerobic conditions and demonstrated a prominent capacity for sucrose re-synthesis that was undetectable under hypoxia. Glucose abundance had distinctive impacts on co-expression networks associated with ANPs, altering network partners and aiding persistence of interacting networks under prolonged hypoxia. Among the ANP networks, two highly interconnected clusters of genes formed around Pyruvate decarboxylase 3 and Glyceraldehyde-3-phosphate dehydrogenase 4. Genes in these clusters shared a small set of cis-regulatory elements, two of which typified glucose induction. Collective results demonstrate specific, previously unrecognized roles of sugars in low-oxygen responses, extending from accelerated onset of initial adaptive phases by starvation stress to maintenance and modulation of co-expression relationships by carbohydrate availability

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Riboflavin (vitamin B(2)) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction