Nitrogen assimilation in crops (II)

1. Nitrogen assimilation in crops (II) March 25, 2011

2. OUTLINE • Introduction of model legume plant and rhizobacterium. • Nodule formation (organogenesis) Process. • Genome sequencing and gene function of rhizobacterium. • Host specificity of rhizobacterium. • Nod-factor-signaling pathway. • Legume nodule senescence.

3. Grain and forage legumes are grown on some 180 million Ha, or 12% to 15% of the Earth’s arable surface. They account for 27% of the world’s primary crop production, with grain legumes alone contributing 33% of the dietary protein nitrogen (N) needs of humans. • 2. Legumes (predominantly soybean and peanut [Arachis hypogeae]) provide • more than 35% of the world’s processed vegetable oil, and soybean and • peanut are also rich sources of dietary protein for the chicken and pork • industries.

4. Major research areas supporting improved food and feed as a major goal of cross-legume genomics Legume family: soybeans, peas, beans, clovers etc.

5. Introduction 1. Root nodules are organs induced on most species of legume plants by symbiotic, N2-fixing bacteria of the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium, collectively called Rhizobia (根瘤菌屬). 2. Medicago truncatula (蒺藜苜蓿) and Lotus. japonicus(百脈根) are being used as a model system to study indeterminate-type and determinate type nodules, respectively. 3. Some plants are naturally able to acquire nitrogen from the air through a process called symbiotic nitrogen fixation. In broad terms, this process requires a close interaction between a soil bacterium, Rhizobium, and the roots of plants of the legume family. 4. Plant host: root-hair curling, formation of an infection thread by the bacterium, nodule initiation, control of nodule number, and nitrogen fixation itself. Lotus. japonicus Medicago truncatula

6. 根瘤菌的分類 根瘤菌自1889 年分離出來，至今已有一百多年，根瘤菌的分類 在早期是以宿主植物為依據，之後發現同一宿主之菌種有快生 及慢生型的世代時間不同，開始發現菌種間之不同 。近年來分 子生物學分析的進步，在微生物的分類上有了新的發展，主 要是利用16S rDNA 及DNA-DNA 雜合的差異為重點，及配合 其他生化分析的分類法，於是新種之分類一一出現。根瘤菌的 分類正在蓬勃發展中，至今根瘤菌在變形菌門(Proteobacteria) 中α 型至少九屬(包括Rhizobium, Allorhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Blastobacter, Azorhizobium, Devosia 及 Methylobacterium)及β 型 有二屬(包括Burkholderia 及Ralstonia)。

9. 5. Bacterials: complete genome sequencing, revealed all the bacterial genes needed for both nodulation (Nod and Nol genes) and nitrogen fixation (Fix genes). 6. plant-microbe interaction or symbiotic nitrogen fixation (SNF): the plant provides its beneficial endosymbiont with photosynthate, together with other nutrients, in exchange for valuable fixed nitrogen, in the form of ammonium and amino acids. 7. Plant (host) and bacterials interaction: flavonoid-Nod factor (nodulation factors, lipo-chito-oligosaccharides). The nature of both the flavonoid signal and the structure of Nod factor are central to the maintenance of specificity in this interaction.

10. Nitrate assimilation Nitrogen assimilation

11. 所有的微生物固氮皆因具有固氮酵素(nitrogenase)之作用能力，固氮酵素在根瘤內或在游離的微生物內都受到特殊的保護，避免受氧氣的破壞。更特別的是固氮酵素能還原多種反應物，能還原氮氣(N2)轉化成氨(NH3 ) 外，尚能還原乙炔( C 2H 2 ) 成乙稀(C2H4)，氰(CN-)還原成氨(NH3)及甲烷(CH4)等的工作，此類物均含 N ≡N、N≡C或C≡C之物質，如表二所示，而對C-≡O+則無法作用。

12. Nitrogenase complex and nodule formation in pea 64 Kda Homo- dimer 240Kda heterotetramer - 400 mV

13. The mechanism of nitrogenase turnover Journal of Integrative Plant Biology 2008, 50 (7): 786–798 In nitrogen-fixing bacteria, nitrogenase is encoded by a set of operons which includes regulatory genes (such as nifLA), structural genes (such as nifHDK) and other supplementary genes.

14. Temperate legume: Alfalfa, pea Amides: glutamine and asparagine

15. Tropical legumes: Soybean (glycine), cowpeas (vigna) Ureides: Allantoin, Allantoic acid

18. Trends in Microbiology 14 (4): 161-168 (2006)

19. Polyhydroxybutyrate (PHB) accumulates in large amounts in free- living and bacteroid cells of manyspecies of rhizobia, but the significance of this reserve material for nitrogen fixation is still unclear. PHB has been suggested to provide reserves of carbon and reductant that are important in sustaining respiratory activities to protect nitrogenase from damage by and in extending nitrogen fixation into the pod-filling stage. On the other hand, there is evidence that suggests that, at least in some species of bacteroids, PHB reserves are not important for nitrogen fixation, and may actually reduce its capacity. Bacteroids in some symbioses (eg, chickpea, alfalfa) do not accumulate PHB, and nodules formed with Rhizobium mutants unable to synthesise PHB exhibit increased nitrogen-fixing activity. Indeed, seeds from Phaseolus vulgaris plants nodulated by one such R. etli mutant contained significantly more nitrogen than seeds from plants nodulated with the wild type strain.

22. Two types of nodules • indeterminate, generally elicited on temperate legumes, such as Medicago sativa, Vicia hirsuta, and . • determinate, generally found on tropical legumes, such as Glycine max, L. japonicus, and Phaseolus vulgaris, the type and size being determined by the host plant .

23. pea Soybean New phytologist (2005) 165: 683-701

24. Nodule formation (organogenesis) Process (1) root hair deformation. (2) dedifferentiation of specific root cortex cells. (3) development of a transient structure called the nodule primordium, formed by different cell types. (4) bacterial invasion either by intercellular growth, or by growth inside newly formed, specialized tubular structures, the infection threads (ITs). (5) generation of a nodule-specific meristem, or foci of meristematic activity. (6) differentiation of the nodular tissues, namely the outer cortex, endodermis, and inner cortex (or parenchyma), containing the vascular strands, and the central tissue made up of cells invaded by rhizobia (invaded cells, ICs) and uninvaded cells (UCs). (7) growth. (8) senescence.

29. The Rhizobium infection process Current Opinion in Plant Biology 9: 110-121 (2006)

35. Current Biology Vol 15 No 6 R196 (2005)

37. 已經確定了超過50個結瘤基因，命名了nodA至nodX一因此，又繼續用nol和noe表示結瘤基因。這些基因在不同的菌株中排列順序和所處的基因簇均不一樣。根據不同的結瘤基因突變體對結瘤過程的影響，可將結瘤基因分為3類，一是共同結瘤基因，如，nodABDIJ,由於共同結瘤基因突變或缺失造成根瘤菌喪失結瘤的功能，可由種間的共同結瘤基因互補得以恢復。nodABC產生使根毛變形的物質，並參與植物早期結瘤素的表達。大多數根瘤菌的nodABC處於同一操縱子中，nodIJ一般位於nodC的下遊。nodIJ突變在R. leguminosarun；中表現結瘤推遲。nodABDIJ在4個根瘤菌屬中均有，認為這4個屬的起源是相同的，二是寄主專一性基因，如，nodEFL, nodMNT, nodO，這類基因的突變一般僅會導致延遲結瘤和結瘤量少，或者寄主範圍發生變化，不同種的根瘤菌間堿基同源性差，其突變體不能被種間互補。第三類是結瘤的調節基因，主要是，nodD基因，過去將其歸為共同結瘤基因，因為4個屬的根瘤菌中均具有nodD基因，且具有一定的同源性，但不同的菌株的nodD的拷貝數不同。nodD基因編碼正向轉錄調節蛋白質，是一種其他nod基因表達所必需的蛋白質。在豌豆根瘤菌中nodD又受其自身負反饋調節，NodD蛋白可與，nod box DNA形成專一的核酸一蛋白質複合物、說明它是一個能結合DNA的轉錄啟動因子。

41. Nod factors induce different molecular,physiological, and morphological host plant responses (1) changes in the intracellular concentration of Ca+2. (2) the production of a nonflavonoid compound causing a further increase in the induction of the nod genes . (3) the expression of chalcone synthase, a key enzyme in the biosynthesis of flavonoids. (4) the expression of chitinases that degrade Nod factors, thus controlling their concentration, or selectively destroying nonspecific factors. (5) changes in plant hormone balance. (6) the generation of reactive oxygen species.

45. ①不同根瘤菌的结瘤因子通常不同。②一种根瘤菌可以产生许多种结瘤因子①不同根瘤菌的结瘤因子通常不同。②一种根瘤菌可以产生许多种结瘤因子

47. Activation of the Nod factor signaling and entry receptor complexes Current Opinion in Plant Biology 9: 110-121 (2006)

49. Genetics and functional genomics of legume nodulation Current Opinion in Plant Biology 9: 110-121 (2006)

50. The postulated position and interaction between elements of the Nod signal recognition pathway Current Opinion in Plant Biology 9: 110-121 (2006) Current Opinion in Plant Biology 8: 346-352 (2005)