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provided by Elsevier - Publisher Connector Current Biology, Vol. 15, 531–535, March 29, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.01.042 Symbiotic Leghemoglobins Are Crucial for in Root Nodules but Not for General Plant Growth and Development

Thomas Ott, Joost T. van Dongen, Catrin Gu¨ nther, moglobin genes are expressed at very low levels in Lene Krusell, Guilhem Desbrosses,1 nodules (see http://www.tigr.org/). Expression of multi- Helene Vigeolas, Vivien Bock, Tomasz Czechowski, ple symbiotic leghemoglobin genes in nodules has sty- Peter Geigenberger, and Michael K. Udvardi* mied attempts to use classical forward genetics to ad- Max Planck Institute of Molecular Plant Physiology dress the physiological role(s) of the corresponding Am Mühlenberg 1 during symbiotic nitrogen fixation (SNF). How- 14476 Golm ever, the high degree of DNA sequence identity be- Germany tween the three symbiotic leghemoglobin genes of L. japonicus (Figure 1B) enabled us to take a reverse- genetics approach to eliminate the expression of all Summary three genes simultaneously. Using RNA interference (RNAi), we succeeded in reducing the levels of symbi- are ubiquitous in nature and among the otic leghemoglobin mRNA in two of five independent, best-characterized proteins [1–9]. Genetics has re- stable transgenic lines of L. japonicus. Expression of all vealed crucial roles for human hemoglobins [10], but three symbiotic leghemoglobins was practically elimi- similar data are lacking for plants. Plants contain nated in these two LbRNAi lines: Transcript levels of symbiotic and nonsymbiotic hemoglobins [11]; the all three genes were below 3% of those in wild-type former are thought to be important for symbiotic ni- nodulated roots three weeks after inoculation with rhi- trogen fixation (SNF). In , SNF occurs in spe- zobia (Figure 2A). In contrast, transcript levels of the cialized organs, called nodules, which contain mil- two nonsymbiotic leghemoglobin genes, LjNSG1 and lions of nitrogen-fixing , called bacteroids [12, LjNSG2, were only slightly reduced in the LbRNAi lines 13]. The induction of nodule-specific plant genes, in- in comparison to in the wild-type controls (Figure 2A). cluding those encoding symbiotic leghemoglobins Leghemoglobin was undetectable in LbRNAi (Lb), accompanies nodule development [14, 15]. Leg- nodules by Western-blot analysis (Figures 2A and 2B), hemoglobins accumulate to millimolar concentrations and intact nodules on LbRNAi plants were white in- in the cytoplasm of infected plant cells prior to stead of the leghemoglobin-pink of wild-type nodules nitrogen fixation and are thought to buffer free oxy- (Figure 3A). gen in the nanomolar range, avoiding inactivation of The LbRNAi lines lacking leghemoglobin protein al- -labile while maintaining high oxy- lowed us to answer the long-standing question: Are gen flux for respiration [16]. Although widely accepted, these prominent nodulins required for SNF? Under symbiotic conditions in which plants were completely this hypothesis has never been tested in planta. Using dependent upon rhizobia for fixed nitrogen, LbRNAi RNAi, we abolished symbiotic leghemoglobin synthesis lines exhibited the classic symptoms of extreme nitro- in nodules of the model legume Lotus japonicus. This gen limitation: severely stunted growth, an increase in caused an increase in nodule free oxygen, a decrease the root/shoot ratio, and delayed flowering compared in the ATP/ADP ratio, loss of bacterial nitrogenase pro- to the wild-type. They also exhibited yellow chlorotic tein, and absence of SNF. However, LbRNAi plants leaves and anthocyanin accumulation in the stems (Fig- grew normally when fertilized with mineral nitrogen. ure 3B). In contrast, wild-type control plants thrived These data indicate roles for leghemoglobins in oxygen under these conditions. Consistent with their nodule- transport and buffering and prove for the first time that specific expression pattern, the three symbiotic leghe- plant hemoglobins are crucial for symbiotic nitrogen moglobins of L. japonicus were not required for plant fixation. growth and development under nonrestrictive nutrient conditions. LbRNAi lines grew normally and flowered at Results and Discussion the same time as wild-type control plants when grown on soil containing mineral and organic nitrogen (Figure Legume genomes typically contain several symbiotic 3C). Thus, the symbiotic leghemoglobins have evolved and nonsymbiotic leghemoglobin (Lb) genes (Figure a truly -specific set of functions that are re- 1A). For instance, Lotus japonicus contains three sym- quired for SNF. biotic leghemoglobins, LjLb1, LjLb2, and LjLb3, which Although LbRNAi lines were defective in SNF, nod- are expressed exclusively in nodules, and two nonsym- ules developed normally on these plants for the first biotic leghemoglobins, LjNSG1 and LjNSG2 [17]. Ex- three weeks after inoculation. In particular, nodule mor- pressed-sequence-tag (EST) data for L. japonicus indi- phology and the extent of central, infected tissue were cate that the three symbiotic leghemoglobin genes are unchanged in LbRNAi lines compared to the wild-type transcribed at similar levels in nodules and together ac- (Figure 3A). Furthermore, bacterial population density count for more than 10% of all plant messenger RNA was similar in nodules of LbRNAi and wild-type plants: (mRNA) in this organ. In contrast, the nonsymbiotic he- The bacterial density in three-week-old nodules of LbRNAi plants was 87% (±11% standard deviation *Correspondence: [email protected] (SD),n=6)that of the wild-type nodules, as measured 1Present address: Université Montpellier 2, CC 002, Place Eugène by real-time polymerase chain reaction (PCR) of nodule Bataillon, F-34095 Montpellier Cedex 05, France. DNA; primers to the bacterial genes nifH and sigA were Current Biology 532

Figure 1. Phylogenetic Tree and Multiple-Sequence Alignment of Hemoglobins from L. japonicus and Other Plant Species Included in the unrooted phylogenetic tree are the Arabidopsis hemoglobins AtHb1 (TIGR TC211680) and AtHb2 (TC199105); the rice hemoglo- bins OsHb1 (TC231324), OsHb2 (TC224250), OsHb3 (TC229216), and OsHb4 (TC246382); the Medicago hemoglobins MtLb2 (TC76879), MtTC76878, MtTC76551, MtLb29 (TC77993), MtTC76987, MtTC80495, and MtGLB1 (TC76971); and the L. japonicus hemoglobins LjLb1 (AB042716), LjLb2 (AB042717), LjLb3 (AB008224), LjNSG1 (TC16046), and LjNSG2 (TC10713). Phylogenetic analyses based on protein se- quence (A) and DNA-sequence alignment (B) were performed via the GenomeNet CLUSTALW server (Koyoto Centre: http://clustalw. genome.jp). The coding sequence of LjLb2 was used for RNAi experiments. used. Differentiation of rhizobia into nitrogen-fixing bac- rhizobia induce expression of nif genes in these nod- teroids involves induction of many genes, including the ules, although nifH transcript levels were lower than in nitrogenase structural genes, nifHDK. Measurement, nodules of wild-type plants (21 ± 4% of wild-type, n = using real-time reverse-transcription PCR (RT-PCR), of 6). Reduced nifH transcript levels in LbRNAi nodules nifH transcript levels in LbRNAi nodules showed that may have resulted from increased levels of free oxygen Symbiotic Nitrogen Fixation Requires Leghemoglobin 533

Figure 3. Symbiotic and Nonsymbiotic Phenotypes of LbRNAi Lines (A) Typical nodules of wild-type and LbRNAi plants 14 days after inoculation with rhizobia. The top two images show whole and sec- tioned wild-type nodules, respectively, and the bottom pair depicts the same for LbRNAi line 3-2. Note the absence of leghemoglobin pigmentation in the LbRNAi nodule but the similar extent of dark- staining, infected cells in the central zone of the nodule section. The scale bars indicate 1 mm. (B) Ten-week-old rhizobia-inoculated plants. Three individuals of two independent LbRNAi lines (2-1, 2-2, 2-3 and 3-1, 3-2, 3-3) are compared with a typical wild-type control (far left). Plants were Figure 2. Molecular Characteristics of LbRNAi Lines grown in sand without added mineral nitrogen. (A) Leghemoglobin transcript levels in nodulated roots 3 weeks af- (C) Ten-week-old noninoculated plants grown in soil with nitrogen ter inoculation of wild-type (left, in black) and LbRNAi lines 2-2 fertilizer. Two LbRNAi individuals are compared to a typical wild- (middle) and 3-2 (right). Transcript levels are expressed relative to type plant (left). those of the constitutive gene ubiquitin [21]. The inset depicts Western-blot detection of leghemoglobins in isolated nodules of the wild-type (wt) but not of the two LbRNAi lines (2-2 and 3-2). ent, with more than 50% of ambient oxygen at 0.2 di- Lane M shows markers of 14 and 18 kDaA. (B) Western-blot detection of leghemoglobins (Lb, above) and ni- ameters and never less than 4.5%, even at the center trogenase NifD (below) in nodulated roots of wild-type (wt) and of nodules (Figure 4). Thus, leghemoglobins help to es- LbRNAi line 3-2, from 7 to 21 days post-inoculation (dpi). Western- tablish very low free-oxygen concentrations throughout blot analyses were performed after SDS-PAGE of 20 ␮g protein. much of a wild-type nodule. Interestingly, despite the increase in free oxygen for oxidative phosphorylation, in these nodules (see below). Expression of nif genes in rhizobia is negatively regulated by oxygen [18, 19]. Despite the presence of nif gene transcripts in LbRNAi nodules, NifD protein was undetectable by Western- blot analysis, in contrast to its abundance in wild-type nodules (Figure 2B). This suggests that nitrogenase proteins are unstable in LbRNAi nodules, and that this instability accounts for the phenotype of symbiotic LbRNAi plants (Figure 3B). The elimination of leghemoglobin protein in LbRNAi lines enabled us to test, for the first time ever, the phys- iological role of these proteins in nodules. Using a nee- dle-type fiberoptic oxygen microsensor, we found that steady-state levels of free oxygen throughout nodules were higher for the LbRNAi lines than for wild-type con- trols (Figure 4). Wild-type nodules exhibited a steep Figure 4. Transects of Free Oxygen in Nodules of Wild-Type and oxygen gradient from the surface toward the center of LbRNAi Lines the nodule, with levels less than 20% of ambient oxy- Oxygen levels are expressed as a percentage of the concentration gen within 0.2 diameters of the surface and below the in air, and the surface and center of nodules are indicated on the x-axis by 0.0 and 0.5, respectively. Wild-type is represented by limit of detection (1% ambient oxygen) for much of the filled circles and LbRNAi lines are represented by open circles. central, infected zone (Figure 4). In contrast, nodules Data points represent mean ±SD of between 11 and 13 indepen- from LbRNAi lines exhibited a shallower oxygen gradi- dent measurements. Current Biology 534

and despite the loss of a major sink for ATP in the form GATEWAY cassette from vector pJawohl8, obtained from Imre of bacteroid nitrogenase in nodules of LbRNAi lines, the Somssich) was used as a binary vector containing a kanamycin- nodule ATP/ADP ratio was more than 4-fold lower in resistance gene for selection of plant transformants. Lotus japoni- cus plants were transformed as described previously [22]. these lines than in the wild-type. The concentrations of ATP and ADP in LbRNAi nodules were 56.9 ± 10.9 and 105.4 ± 26.3 nmol/g fresh weight (±SD, n = 3), respec- RNA Extraction and Real-Time RT-PCR RNA was extracted from 100 mg plant tissue with the RNeasy Kit tively, whereas in wild-type nodules the corresponding (QIAGEN). DNaseI treatment of 1 ␮g total RNA, cDNA synthesis, values were 97.8 ± 15.7 and 39.9 ± 10.5 nmol/g fresh and real-time RT-PCR were carried out as described previously weight, respectively. Despite the reduced cellular en- [23]. The following primers were used to amplify the different leghe- ergy status (ATP/ADP) in LbRNAi lines compared to that moglobins: in the wild-type, the total amount of ATP plus ADP was not significantly different between the two types of LjLb1 nodules. Thus, high concentrations of leghemoglobins forward: 5#-TTTGAGCACTGCTTGGGGAGTAGCT-3# enhance energy metabolism in nodule cells, presuma- reverse: 5#-CTGCAATTAAGAAGGCAATG-3# bly by increasing the overall amount of oxygen (free LjLb2 # # plus protein bound) and thereby the flux of oxygen to forward: 5 -ATTGAGCACTGCTTGGGGAGTAGCC-3 reverse: 5#-CTGCAATTAAGAAGGCAATG-3# sites of respiration. LjLb3 The results presented here have positive implications forward: 5#-CTTGAGCACCGCTTGGGAAGGAGCA-3# for the functional analysis of other nodulins. We have reverse: 5#-CTGCAATTAAGAAGGCAATG-3# demonstrated that RNAi is a potent way to simulta- neously reduce or eliminate transcripts and proteins Plant-gene transcript levels were normalized to those of ubiquitin, derived from multiple highly expressed homologous as described previously [21]. After reverse transcription of nodule RNA in the presence of 1 pM of the primers FeNif1R and Mlsig- genes in legumes. Thus, in the future, RNAi should help Ar1, we measured rhizobial nifH and sigA transcript levels by using to resolve the symbiotic functions of other nodulins, the following PCR primers: many of which are members of multigene families. In summary, we have shown that the most prominent nifH of legume nodulins, the leghemoglobins, are required forward: 5#-TCCAAGCTCATCCACTTCGTG-3# for SNF, but not for plant growth and development in reverse: 5#-AGTCCGGCGCATACTGGATTA-3# the presence of an external source of fixed nitrogen. sigA Physiological analysis of nodules from LbRNAi plants forward: 5#-GCCCTCTGCTCGACCTTTCC-3# revealed the crucial contribution of leghemoglobins to reverse: 5#-AGCATCGCCATCGTGTCCTC-3# establishing low free-oxygen concentrations but high Levels of nifH transcript were normalized to those of the constitu- energy status in nodules, conditions that are necessary tive gene, sigA. for effective SNF. This work represents the first demon- stration of a role for hemoglobins in plants and helps DNA Extraction and Real-Time-PCR Quantification to close the gap in the understanding of DNA was isolated from nodules via a published protocol [24]. Plant functions in plants versus animals. genomic DNA was quantified with primers to ubiquitin under stan- dard real-time-PCR-reaction conditions [21, 23]. Rhizobial DNA Experimental Procedures was quantified with the primers to nifH and sigA, described above.

Plant Cultivation Lotus japonicus ecotype GIFU (B-129) seeds were scarified in 98% Immunoblotting sulfuric acid for 15 min, sterilized in 2% sodium hypochlorite con- Twenty milligrams of nodulated roots were homogenized in a buffer taining 0.01% Tween 20 for 15 min, rinsed five times with sterile containing 10 mM Tris (pH 7.5), 140 mM NaCl, 5 mM Ethylenediami- distilled water, and germinated on moist, sterile filter paper for 3 netetraacetic acid (EDTA), 1% Triton X-100, 1 mM Phenylmethylsul- days. Germinated seedlings were transferred to agarose plates phonylfluoride (PMSF), and 1 mM Dithiothreitol (DTT). After cen- trifugation for 10 min in a microcentrifuge, supernatant-protein containing 25% B&D medium [20] and 0.1 mM KNO3. Plants were grown in a 16/8 hr day/night cycle with a light intensity of 150 concentration was determined via the Bradford method (Biorad). ␮Em−2s−1 at 21°C in a controlled-environment chamber (York In- Extracted protein was separated by SDS-PAGE on a commercial ternational, model P-U201AF). Plants were inoculated at 2 weeks 10% Bis-Tris gel (Invitrogen) according to the manufacturer’s in- of age with a 1:50 dilution of a 2-day-old liquid culture of Mesorhi- structions (100V, 1.5 hr). We blotted proteins onto a Protran® nitro- zobium loti strain NZP2235. Alternatively, plants were grown in cellulose membrane (Schleicher und Schuell) by using the XCellII a greenhouse in coarse quartz sand or soil, as described previ- Blot Module (Invitrogen). Equal loading of lanes and quality of ously [21]. transfer were checked by Ponceau staining of membranes. Antise- rum against leghemoglobin was kindly provided by Norio Suganuma (Aichi University of Education, Japan), and antiserum RNAi Construct and Plant Transformation against the NifD subunit of nitrogenase was provided by Ton Bis- A 401 base pair DNA fragment of LjLb2 (AB042717) was PCR ampli- seling (University of Wageningen, The Netherlands). fied from cDNA via the following primers:

LjLbEntry Oxygen Measurements forward: 5#-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGGTTT We measured free-oxygen concentrations in intact 3-week-old CACTGCACAGCAAG-3# nodules by using a needle-type fiber-optic oxygen microsensor reverse: 5#-GGGGACCACTTTGTACAAGAAAGCTGGGTCATAGG (MicroxTX2, Presens, Regensburg, Germany) with a tip diameter of CTACTCCCCAAGCA-3# less than 50 ␮m[25]. The location of the tip within the nodule was derived from the scaling of the micromanipulator with which the These primers contained recombination sites for GATEWAY cloning sensor was driven through the tissue. The entire procedure was (Invitrogen). pBINAR-RNAi (pBIN19 backbone with a converted performed under a binocular microscope. Symbiotic Nitrogen Fixation Requires Leghemoglobin 535

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