From Model to Crop: Functional Analysis of a STAY-GREENGene in the Model Legume Medicago truncatula andEffective Use of the Gene for Alfalfa Improvement1[W][OA]
Chuanen Zhou2, Lu Han2,3, Catalina Pislariu, Jin Nakashima, Chunxiang Fu, Qingzhen Jiang, Li Quan,Elison B. Blancaflor, Yuhong Tang, Joseph H. Bouton, Michael Udvardi, Guangmin Xia, and Zeng-Yu Wang*
Forage Improvement Division (C.Z., L.H., C.F., Q.J., J.H.B., Z.-Y.W.) and Plant Biology Division (C.P., J.N.,L.Q., E.B.B., Y.T., M.U.), Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401; and Key Laboratoryof Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences,Shandong University, Jinan 250100, China (L.H., G.X.)
Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widelygrown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with thetransposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintainedgreen leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecularanalyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profilinganalysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved indifferent biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence.MtSGRexpression was detected across all nodule developmental zones and was higher in the senescence zone. The number of youngnodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers werereduced in the sgrmutant. Based on theMtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lineswere produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficialtrait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of thetransgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content.This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an importantcommercial crop.
Senescence of forage and turf species is of specialinterest because the market value of these widelygrown plants is closely related to the visual appear-ance of their foliage. The most obvious phenomenon ofplant senescence is leaf color change from green toyellow or red. Color change of leaves during senes-cence is caused by chlorophyll degradation, combinedwith carotenoid retention or anthocyanin accumula-tion (Park et al., 2007). Higher plants contain two typesof chlorophylls, chlorophyll a (Chl a) and chlorophyll b(Chl b). The conversion of Chl b to Chl a is consideredthe first step of chlorophyll degradation (Hortensteiner,2009). Chl a is continuously converted by multistep
pathways to linear colorless and nonfluorescent tetra-pyrroles (Hortensteiner, 2006).
stay-green (sgr) mutants with visible retention ofchlorophyll have been identified from different plantspecies (Park et al., 2007; Ren et al., 2007; Alos et al.,2008; Sato et al., 2009). They have been classified intofive types, A, B, C, D, and E, based on chlorophyllbehavior during senescence (Thomas and Howarth,2000). The most extensively investigated mutant istype C, in which chlorophylls are retained in senescentleaves while their photosynthetic competence de-creases. This type of mutant was first found in a foragecrop, meadow fescue (Festuca pratensis), and namedsenescence-induced degradation (Thomas and Stoddart,1975; Thomas, 1987; Armstead et al., 2007). In recentyears, type C stay-green-related genes have beenidentified in Arabidopsis (Arabidopsis thaliana), rice(Oryza sativa), pea (Pisum sativum), and other speciesand have been named SGR (Jiang et al., 2007; Parket al., 2007), NONYELLOWING (NYE1; Ren et al.,2007), GREEN-FLESH (GF), or CHLOROPHYLL RE-TAINER (Barry et al., 2008). Moreover, it has beenshown that SGR cosegregates with the I locus control-ling the green cotyledon trait in pea (Armstead et al.,2007; Sato et al., 2007), which was used as one of theseven traits by Gregor Mendel to establish his famouslaws of genetic inheritance. SGR plays an important
1 This work was supported by the Samuel Roberts Noble Foun-dation.
2 These authors contributed equally to the article.3 Present address: School of Medical and Life Science, University
of Jinan, Jinan 250022, China.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Zeng-Yu Wang ([email protected]).
[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-
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role in the dismantling of chlorophyll protein com-plexes during senescence (Park et al., 2007; Aubryet al., 2008). However, the exact function and mecha-nism of this family of SGR genes have not yet beenelucidated.
Alfalfa (Medicago sativa) is widely cultivated inNorth America and is the third largest crop econom-ically after corn (Zea mays) and soybean (Glycine max)in the United States (Bouton, 2007). Alfalfa is oftenreferred to as “the queen of forage crops” because it ishighly productive, drought tolerant, and provideshigh-quality forage over a long period of time. Mostalfalfa is used for hay production. A key trait for theevaluation of hay value at the market is the color ofalfalfa leaves. Bright green, instead of yellowish look-ing, is one of the physical characteristics of premiumhay.
Alfalfa is an obligate outcrossing and tetraploidspecies (2n = 4x = 32); such features make genetic andgenomic studies difficult and promoted the develop-ment of Medicago truncatula as a model legume. M.truncatula is a diploid, self-fertile species with a shortlife cycle and a small genome. Many tools and re-sources have been developed in M. truncatula, such asretrotransposon and fast-neutron mutant populations(Tadege et al., 2008), ecotype collections (Cook, 1999),EST and genespace sequencing information (Younget al., 2005; Young and Udvardi, 2009), and the GeneExpression Atlas (Benedito et al., 2008).
Like other legume species, alfalfa and M. truncatulahave the ability to respond to nitrogen limitation byestablishing symbiotic interactions with soil bacteria,collectively known as rhizobia, which are able toreduce atmospheric nitrogen (N2) to ammonium.Upon successful entry, rhizobia are hosted insidenew organs, root nodules, where low oxygen levelsallow the nitrogenase enzyme complex to carry outnitrogen fixation. When nodule cells senesce, nitrogenfixation ceases and the symbiotic relationship is lost. Ithas been demonstrated that nodule senescence is acomplicated but regulated process, although themechanism has not been elucidated. It is consideredthat crop yield and seed quality may benefit fromdelayed nodule senescence and the concomitant ex-tended nitrogen fixation (Van de Velde et al., 2006).
In this study, we describe the identification andcharacterization of a sgrmutant from theM. truncatulapopulation tagged with the transposable element oftobacco (Nicotiana tabacum) cell type1 (Tnt1). Geneticand molecular analyses revealed that the mutationwas caused by Tnt1 insertion in the SGR gene ofM. truncatula (MtSGR). Further analyses showed thatSGR is involved not only in leaf senescence but alsoin nodule senescence. RNA interference (RNAi) wasemployed to silence the expression of the endoge-nous SGR gene (MsSGR) in alfalfa. The transgenicalfalfa plants retained much of their chlorophyllduring senescence, stayed green during the naturaldrying process, and showed increased crude proteincontent.
RESULTS
Identification and Phenotypic Characterization of aM. truncatula sgr Mutant during Natural and
Dark-Induced Senescence
A mutant line, NF2089, caught our attention whilescreening large numbers (more than 10,000 lines) ofthe M. truncatula Tnt1 retrotransposon-tagged mu-tants. During natural senescence, the basal leaves ofthe wild type (ecotype R108) turned yellow firstand the whole plant gradually became yellowish.However, the basal leaves of NF2089 did not showsenescent yellow color in the same way as those ofwild-type plants (Fig. 1A). Moreover, the whole plantremained green even after the leaves died (Fig. 1B).Interestingly, the stay-green phenomenon was evidentnot only in leaves but also in other organs, such asanthers and central carpels (Fig. 1C), mature pods (Fig.1D), and seeds (Fig. 1E).
To determine if the NF2089 mutant exhibits a stablestay-green phenotype during dark-induced senes-cence, detached leaves of the mutant and wild typewere placed in darkness for up to 10 d. The leaves ofthe mutant remained green on the 5th d after darknessand turned to light green after 10 d of dark treatment.In contrast, wild-type leaves became light green on the5th d after darkness and turned yellow after 10 d ofdark treatment (Fig. 2A).
To determine chlorophyll content and its possibleimpacts on leaf photosynthesis in the mutant, wholeplants of the mutant and the wild type were trans-ferred to a dark growth chamber. Much of the chloro-phyll was retained in the mutant after dark treatment(Fig. 2B), with 43% of Chl a and 82% of Chl b retainedafter 10 d of dark treatment (Fig. 2C). The ratio of Chla to Chl b in the mutant decreased at 10 d after darktreatment compared with that of the wild type (Fig.2D). The maximal photochemical efficiency (Fv/Fm) ofPSII, an important parameter of PSII activity, was alsomeasured (Fig. 2E). The beginning Fv/Fm value beforedark treatment was similar in both NF2089 and thewild type; while the value of the mutant was slightlyhigher than that of the wild-type after 5 d of darktreatment, the difference in Fv/Fm between the mutantand the wild type was not significant after 10 d ofdarkness. These results indicate that Fv/Fm in themutant decreased in a similar way as the wild typeduring senescence, although the decrease in the mu-tant was slightly slower than that of the wild type.
Chloroplast Structure in Senescent Leaves
The ultrastructure of chloroplasts in both the wildtype and the NF2089 mutant was examined usingtransmission electron microscopy. Under normalgrowth conditions, the structure of chloroplasts inthe NF2089 mutant was similar to that in the wild type,both containing well-formed grana stacks along withdistinct starch granules (Fig. 3, A, B, E, and F). After10 d of dark treatment, clear differences between
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chloroplast structures of the wild type (Fig. 3, C and D)and the NF2089 mutant (Fig. 3, G and H) were ob-served. One difference was the large number ofplastoglobules formed in the wild type, whereas onlya few were observed in the mutant. Moreover, thestructures of grana stacks became obscure in the wildtype, while thick and wide grana stacks were stilldetected in chloroplasts of the NF2089 mutant. Theseresults indicate that chloroplast decomposition in theNF2089 mutant was much slower than that in the wildtype.
Molecular Cloning and Characterization of the SGRGenein M. truncatula
To determine if the stay-green phenotype in NF2089was caused by the mutation of a single gene, themutant was crossed with the wild type and segrega-tion analysis was performed in the progeny. The F1plants did not show the stay-green phenotype whendetached leaves were incubated in darkness. Segrega-tion was observed in F1 seeds and F2 plants. The ratioof green versus yellow F1 seeds was close to 1:3(148:457), and the ratio between stay-green F2 plantsand non-stay-green F2 plants was approximately 1:3(46:143; Supplemental Table S1). Furthermore, all theplants from the green seeds showed the stay-greenphenotype after dark treatment, while the plants fromyellow seeds did not show the phenotype. Theseresults demonstrate that NF2089 is a recessive mutantand that the mutation was caused by the loss offunction of a single gene. They also suggest that thestay-green phenotype in the leaves is associated withthe green color of the seeds.
To determine which defective gene caused the stay-green phenotype in the mutant, thermal asymmetricinterlaced PCR was performed to recover the flankingsequences of the Tnt1 retrotransposon in NF2089. Intotal, 13 retrotransposon insertions at different siteswere recovered from the mutant. One of the insertions(NF2089-9) was found to segregate with the mutantphenotype, based on PCR genotyping (SupplementalTable S2). BLASTn analysis of the sequence flankingthe Tnt1 insertion against theM. truncatulaGene Indexdatabase showed that NF2089-9 was located in a genecorresponding to TC126805. By reverse transcription(RT)-PCR and sequence analysis, the full-length cod-ing sequence (792 bp) was obtained, which showed87% identity with PsSGR (Sato et al., 2007), 76%identity with AtNYE1 (Ren et al., 2007), and 65%identity with rice SGR (Park et al., 2007). Sequenceanalysis of genomic DNA revealed that the gene,designated MtSGR, consists of four exons and threeintrons (Fig. 4A). PCR amplification of MtSGR fromgenomic DNA of the wild type and NF2089 confirmedthat one 5.3-kb Tnt1 was inserted into this gene inNF2089 (Fig. 4B); therefore, the expression of MtSGRwas abolished in the mutant (Fig. 4C). Analysis oftranscription levels of MtSGR in wild-type plantsshowed that the expression of MtSGR was up-regu-lated during dark-induced senescence (Fig. 4D).
Two other Tnt1 mutants with insertions in MtSGR,NF6817 and NF8082, were obtained via a reversegenetics approach. The insertion sites in MtSGR ofNF6817 and NF8082 were in the second intron andthe promoter region, respectively (Fig. 4A). Homozy-gous NF6817 and NF8082 plants exhibited similar
Figure 1. Phenotypic characterizationof the Tnt1-tagged M. truncatulamutant line NF2089. A, Natural senes-cence of leaves at the bottom of wild-type and NF2089 plants after 60 d ofgrowth. B, Natural senescence of wild-type and NF2089 plants. C, Colorchange in mature anthers and centralcarpels of wild-type and NF2089plants. D, Mature pods of wild-typeand NF2089 plants. E, Mature seeds ofwild-type and NF2089 plants.
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phenotypes as the wild type, indicating that the Tnt1insertions of these lines did not impair MtSGR genefunction.
The expression pattern of MtSGR in wild-typeplants was analyzed by utilizing the M. truncatulaGene Expression Atlas (Benedito et al., 2008). Theexpression of MtSGR was detected in almost all theorgans at various developmental stages, with higherlevels in mature seeds and senescing nodules andrelatively low levels in flowers, petioles, stems, pods,
vegetative buds, leaves, roots, and young seeds (Sup-plemental Fig. S1).
Global Gene Expression Profiling of the NF2089 Mutant
To investigate if MtSGR affected downstream genesduring dark-induced leaf senescence in M. truncatula,a microarray analysis was performed using the Affy-metrix Medicago Genome Arrays. After 5 d of darktreatment, fully expanded leaves of both the NF2089mutant and a corresponding control were used toisolate RNA for chip analysis. Compared with thecontrol, 347 genes were repressed, and 1,251 geneswere induced significantly by at least 2-fold in theNF2089 mutant (Supplemental Table S3). These datasuggest that MtSGR probably mostly plays inhibitoryroles in gene expression. The expression levels of 12putative senescence-associated genes were changed,and all of them were up-regulated in NF2089 (Sup-plemental Table S4). Some of up-regulated genes arepredicted to participate in the processes of cellularprotein complex assembly, protein polymerization,and protein modification by small protein conjugationor removal (Supplemental Fig. S2), supporting theview that SGR is involved in the disassembly processof the light-harvesting chlorophyll-binding proteincomplexes (Park et al., 2007; Borovsky and Paran,2008). On the other hand, the expression levels of M.truncatula homologs of chlorophyllase, pheide a oxy-genase, and red chlorophyll catabolite reductase didnot show any obvious changes in the mutant, indicat-ing that the absence of MtSGR does not affect theexpression of these genes in the chlorophyll degrada-tion pathway.
Only 2.5% of up- or down-regulated genes (40 of1,598) encode chloroplast-related proteins (Supple-mental Table S5). This result implies that MtSGRmay participate in broad biological processes duringleaf senescence besides chlorophyll degradation. GeneOntology (GO) analysis was performed using the GOEnrichment Analysis Software Toolkit (Zheng andWang, 2008). The results showed that 168 GO classesand 49 GO classes were enriched, respectively, in up-regulated and down-regulated genes in the NF2089mutant. The representations of probe sets in theenriched GO classes were examined with WEGO (Yeet al., 2006), and the number of genes up-regulated ordown-regulated in each category was calculated (Sup-plemental Fig. S2). Different biological processes, cel-lular components, and molecular functions werefound to be substantially affected in senescing leaveswhenMtSGRwas absent (Supplemental Table S6). Theinvolvement of multiple function categories suggeststhat MtSGR plays a broad role in plant developmentand senescence.
The Involvement of MtSGR in Nodule Senescence
The above-mentioned Gene Expression Atlas anal-ysis revealed that MtSGR expression is relatively high
Figure 2. Stay-green phenotype and chlorophyll characterization of theNF2089 mutant during dark-induced senescence. A, Color change indetached leaves of wild-type and NF2089 plants during dark-inducedsenescence. B, Chlorophyll degradation during dark incubation. fw,Fresh weight. C, Changes in contents of Chl a and Chl b duringsenescence. Black circles, Chl a; white circles, Chl b. D, Chl a/b ratio ofwild-type and NF2089 plants. E, Change in Fv/Fm values during darkincubation. Error bars indicate SE (n = 3). Numbers (0, 5, and 10)indicate days after dark treatment (DAD).
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in senescing nodules, second only to mature seeds(Supplemental Fig. S1). To test a possible association ofMtSGRwith nodule senescence, nodulated roots of thewild type and the NF2089 mutant were analyzed 4weeks after inoculation with Sinorhizobium melilotiABS7 strain. No obvious phenotypic differences be-tween senescent nodules of the wild type and theNF2089 mutant were observed. Both nodule size andthe extent of the senescence zone, as indicated by thegreenish appearance at the proximal end of the nod-ule, were similar in the wild type and the mutant(Supplemental Fig. S3). Although the number of se-nescent nodules in the NF2089 mutant was similar tothat in the wild type, at closer examination, we found asignificantly higher number of young nodules on themutant roots (P , 0.01; Fig. 5A). These young noduleswere pink with no senescence zone (Supplemental Fig.S3), implying a potential correlation ofMtSGRwith theonset of nodule senescence. Furthermore, the pheno-type of senescent nodules of the mutant was similar tothat of the wild type, suggesting that other factors mayalso be involved in the process of nodule senescence.
To study the relationship between MtSGR expres-sion and nodule senescence, quantitative RT-PCRwas used to analyze gene expression in nodulesduring natural senescence and NO3-induced senes-cence inM. truncatula. The relative expression level ofMtSGR was measured in young nodules and natu-rally senescing nodules harvested 4 weeks postinoc-ulation with rhizobia. MtSGR transcript levels innaturally senescing nodules was 2-fold higher thanin young nodules (Fig. 5B). In addition, comparedwith naturally senescing nodules, the MtSGR tran-script level was 8-fold higher in NO3-induced senes-cence (after 5 d of treatment; Fig. 5C).
MtSGR expression was also assessed in wild-typenodules along five functional zones corresponding towell-defined stages of rhizobial infection and noduledevelopment. These zones are as follows: meristem;the invasion zone, where rhizobia are released from thetube-like structures called infection threads into thehost cell cytoplasm and are surrounded by plant cell-derived membranes to form a new organelle, thesymbiosome; interzone II-III, where rhizobia mature
Figure 3. Ultrastructure of chloro-plasts in the wild type (A–D) and theNF2089 mutant (E–H). Chloroplaststructures observed using transmissionelectronmicroscopy at 0 d (A, B, E, andF) and 10 d (C, D, G, and H) after darktreatment are shown. The insets showhigher magnification images of chlo-roplast structures corresponding to thewhite squares in the main panels. G,Grana stack; P, plastoglobule; S, starchgranule. Bars = 500 nm.
Figure 4. Molecular cloning of the MtSGR gene. A, Diagram of the MtSGR gene (1,734 bp) structure showing the four exons(blocks), three introns (lines), and positions of Tnt1 insertions. B, PCR amplification of MtSGR from genomic DNA of the wildtype (WT) and NF2089 showing the presence of a 5.3-kb Tnt1 insertion in the mutant. C, RT-PCR showing that transcription ofMtSGRwas interrupted in NF2089. D, Expression ofMtSGR in leaves of the wild type. Numbers (0, 5, and 10) indicate days afterdark treatment (DAD).
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into rod-shaped, nitrogen-fixing bacteroids; the nitro-gen fixation zone, where bacteroids actively reduceatmospheric nitrogen; and the senescence zone, wherenitrogen fixation ceases and both bacteria and plantcells die (Fig. 5D). Quantitative RT-PCR analysisshowed that MtSGR transcripts were present in allnodule zones, with the highest levels in the senescencezone (Fig. 5E).
To confirm that elevated levels ofMtSGR expressionin the senescence zone could link this gene functionwith degenerative processes of nodule senescence, wemeasured the expression of several nodule senescencemarkers in wild-type and NF2089 senescing nodules.These markers were selected on the basis of senes-cence-specific expression from a Medicago AffymetrixGeneChip data set obtained from spatially resolved
nodule zones (C. Pislariu and M. Udvardi, unpub-lished data; Supplemental Fig. S3). Probe set targetdescriptions are listed in Supplemental Table S7.Quantitative RT-PCR analysis showed that seven outof nine markers were significantly down-regulated inthe sgr mutant, while the expression of the other twomarkers was similar in mutant and wild-type senes-cent nodules (Fig. 5F). The most down-regulated are aphosphate transporter (Mtr.15323.1.S1_s_at) and a ba-sic blue copper protein (Mtr.10725.1.S1_at), which maybe involved in phosphate and copper salvage duringsenescence (Miller et al., 1999; Chapin and Jones, 2009).A putative ripening-related protein (Mtr.10361.1.S1_at)and a Cys proteinase (Mtr.43787.1.S1_at) have un-changed relative expression in NF2089 compared withthe wild type (Fig. 5F).
Figure 5. The involvement of MtSGRin nodule senescence ofM. truncatula.A, Nodule number of the wild type andthe NF2089 mutant. The two asterisksindicate a significance level corre-sponding to P , 0.01. B, Expressionlevel of MtSGR in young nodules andsenescing nodules of the wild type. C,Expression level of MtSGR in NO3-induced senescing wild-type nodules.dpi, Days after rhizobial inoculation.D, Different developmental zones ofthe wild-type nodule. E, Expressionlevel of MtSGR in the different devel-opmental zones of the wild-type nod-ule shown in D. F, Expression level ofnodule senescence marker genes insenescing nodules of the wild typeand the NF2089 mutant. Error barsindicate SE (n = 20) in A and SE (n = 3)in B, C, E, and F.
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Taken together, our findings suggest that MtSGR ispossibly involved in certain nodule development andsenescence pathways.
Isolation of an SGR Gene from Alfalfa
An SGR gene was cloned from alfalfa (designatedMsSGR) based on the high sequence similarity be-tween M. truncatula and alfalfa. Analysis of the de-duced amino acid sequence revealed that the MsSGRprotein contains 263 amino acids, with predicted pI of8.716 and molecular mass of 30 kD. Predicted SGRprotein sequences from several species were collectedfrom GenBank and used for phylogenetic analysis(Supplemental Fig. S4A). The phylogenetic trees wererooted using Physcomitrella patens (moss) SGR anddivided into two clades: one belonging to monocoty-ledonous species and the other belonging to dicotyle-donous species. Phylogenetic analysis showed thatMsSGR and MtSGR were closest to each other andwere clustered close to PsSGR (Supplemental Fig.S4A). The MsSGR sequence showed 98% identity toMtSGR, 86% to PsSGR, 78% to GmSGR1, 67% toAtNYE1, and 64% to OsSGR. The SGR family mem-bers share a highly conserved central region but aredivergent at N and C termini. The amino acid Arg-145(Supplemental Fig. S4B, asterisk), at which site theTnt1 was inserted into the mutant NF2089, is aninvariant residue within the SGR family.
Suppression of MsSGR Expression in Alfalfa by RNAi
To suppress the activity of the endogenous MsSGRin alfalfa, an MsSGR-RNAi vector was constructedand introduced into alfalfa plants by Agrobacteriumtumefaciens-mediated transformation. Twenty trans-genic lines were identified through PCR analysis(Fig. 6A). Quantitative RT-PCR analysis revealed thatfive transgenic lines, SGRi-10, SGRi-17, SGRi-21, SGRi-29, and SGRi-39, had SGR transcript levels reduced bymore than 60% when compared with the empty vectorcontrol line (Fig. 6B). These five transgenic alfalfa lineswere used for further analyses.Southern-blot hybridization analysis confirmed
that the transgene was stably integrated in the alfalfagenome and that the regenerated positive lines weretruly independent transformants. Both single-copyand multiple-copy integrations of the transgene wereobserved in the transgenic lines. Transgenic lineSGRi-21 had single-copy integration; SGRi-29 con-tained two copies of the transgene; and SGRi-10,SGRi-17, and SGRi-39 had at least three copies of thetransgene (Fig. 6C).
Leaf Senescence of the MsSGR-RNAi TransgenicAlfalfa Plants
The effects of MsSGR down-regulation on trans-genic alfalfa were analyzed by incubating detachedleaves in darkness to induce senescence. The trans-
genic lines exhibited a stable nonyellowing phenotypeduring a continuous 20-d dark treatment. Althoughthe detached leaves from the transgenics turned lightgreen on the 5th d of dark incubation, the green colorremained until the end of the 20-d treatment (Fig. 7A).In contrast, the empty vector control and wild-typeleaves began to turn yellow by the 5th d, and theyellow color spread gradually to full leaves by the 10thd of dark treatment.
To determine the effects of MsSGR suppression atthe whole plant level, live transgenic plants wereplaced in a dark growth chamber. During naturalsenescence of alfalfa plants, the lower leaves usuallysenesce earlier than do the upper leaves. A similarphenomenon was observed in dark-induced senes-cence in alfalfa. On the 5th d of dark treatment, basalleaves of the control plants began to turn yellow, whilethe leaves of all the transgenic lines remained green.On the 10th d of dark treatment, whole plants of thecontrols became yellowish, with some basal leavesturning red, while the transgenic lines kept the stay-green phenotype, although some of the leaves at thetop became wrinkled. After 15 and 20 d of darktreatment, almost all the leaves in the control becameyellow and red, while the leaves of the transgenic linesremained a similar green color to that of the 10th d ofdark treatment (Fig. 7B).
Transmission electron microscopy analysis re-vealed that chloroplast outline, shape, and contentin the transgenic alfalfa lines showed similar tem-poral changes as did the M. truncatula NF2089 mu-tant during dark-induced senescence (SupplementalFig. S5).
To determine if delayed leaf senescence was associ-ated with the repression of MsSGR expression, thetranscript level of MsSGR was measured in leavesof the wild type and the SGRi-39 line under darktreatment (Supplemental Fig. S6). MsSGR was up-regulated dramatically in leaves of the wild type,especially after 5 and 10 d in darkness. The expressionlevel of MsSGR in SGRi-39 varied slightly and wasconsistently lower than that of the control at differenttime points of the dark treatment.
Under natural growth conditions, as plants grow,the old leaves lose function and drop off the plant. Acolor difference was observed in these fallen leaves,with control leaves yellow while transgenic leavesremained green (Supplemental Fig. S7).
Chlorophyll Content and Physiological Changes inTransgenic Alfalfa during Dark Incubation
Chlorophyll content, Chl a/b ratio, and PSII func-tionality (measured as Fv/Fm) were measured duringdark treatment of living transgenic alfalfa plants. Nodifference was observed in chlorophyll loss after 5 d ofdark treatment (Fig. 8A). Significant changes wereobserved, however, after 10 d of dark treatment. By the20th d of dark treatment, only 5% of chlorophylls
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remained in the control leaves, while more than 50% ofchlorophylls remained in most of the MsSGR-RNAilines (50.6% in SGRi-10, 52.8% in SGRi-21, 55.6% inSGRi-29, and 60.2% in SGRi-39). Among the transgeniclines, SGRi-39 retained the highest level of chloro-phylls (60.2%), which was double the amount ofchlorophylls retained in SGRi-17 (30.0%).
No difference in Chl a/b ratio between transgenicand control plants was observed after 0 and 5 d of darktreatment. However, after 10 and 20 d of darkness, theRNAi lines showed large decreases in Chl a/b ratios(Fig. 8B). As shown in Figure 8C, Fv/Fm values de-
creased similarly during the course of senescence inthe transgenic lines and the control.
Improvement of Forage Quality of MsSGR-RNAiTransgenic Alfalfa
When harvesting alfalfa for hay production, thefresh-cut plants are usually dried and baled in thefield. To evaluate the potential application of MsSGR-RNAi alfalfa, we mimicked the harvest and dryingprocess using these transgenic materials. The trans-genics were greener than the control after 5 d of
Figure 6. Molecular characterization of alfalfaMsSGR-RNAi transgenic lines. A, PCR analysis ofregenerated alfalfa plants together with the pos-itive control (pANDA35HK-SGR plasmid), nega-tive control (Wild-type), and empty vector control(CTRL). The sizes of DNA fragments are 699 bpfor nptII, 375 bp for hph, and 633 bp for guslinker. B, Quantitative RT-PCR analysis of MsSGRgene expression in transgenic lines. All valueswere normalized using the empty vector control.Error bars indicate SE (n = 3). C, Southern-blotanalysis of KpnI-digested genomic DNA fromleaves of the wild type and MsSGR-RNAi trans-genic lines. The DNAwas probed with 699 bp ofgus linker fragment from the pANDA35HK vector.
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drying. This difference in visual quality of the trans-genics and the control became more obvious after 10 dof drying, with transgenics showing a more attractivegreenish color (Fig. 9).Forage nutritive quality analysis revealed that most
of the transgenic lines (except SGRi-17) had increasedcrude protein content (Fig. 10). Compared with thecontrol, the level of increase in crude protein contentof SGRi-29, -21, -10, and -39 varied from 2.3% to 5.5%.Other nutritive quality traits, including in vitro truedry matter digestibility, total digestible nutrients,acid detergent fiber content, neutral detergent fibercontent, magnesium concentration, and phosphorusconcentration, did not show consistent changes be-tween the transgenic lines and the control (Supple-mental Fig. S8).
DISCUSSION
Both M. truncatula Mutant NF2089 and MsSGR-RNAiAlfalfa Show Nonfunctional Type C Stay-Green
Phenotype during Senescence
A stay-green mutant, NF2089, was identifiedthrough screening a M. truncatula mutant collectiongenerated by Tnt1 retrotransposon insertional muta-genesis. The stay-green phenotype is caused by aTnt1 insertion in the MtSGR gene, which is inducedby senescence and responsible for chlorophyll break-down. During both natural and dark-induced senes-cence, the sgrmutant showed the stay-green phenotypenot only in leaves, stems, mature pods, and seeds,as reported in other species (Sato et al., 2007; Aubryet al., 2008), but also in anthers and central carpels,which has not been reported before. In addition, weisolatedMsSGR from alfalfa and produced transgenicplants with an MsSGR-RNAi construct. The trans-genic alfalfa with down-regulated expression ofMsSGRshowed the same stay-green phenotype.
In both the sgr M. truncatula mutant and MsSGR-RNAi transgenic alfalfa, a large portion of Chl a andChl b was retained after senescence, suggesting thatthe SGR gene plays an important role in degradationmechanisms of both Chl a and Chl b in the two species.Although much of the chlorophylls were retained aftersenescence, the Fv/Fm values of the mutant and thetransgenics decreased in a similar manner, as did thoseof the controls. These results indicate that the sgrmutant and MsSGR-RNAi transgenic alfalfa showedthe nonfunctional type C stay-green phenotype duringsenescence.
SGR Is Implicated in Nodule Developmentand Senescence
In indeterminate nodules such as those of M. trun-catula, where an active meristem is permanently main-tained, functional zonation along the nodule is easilydistinguishable. Adjacent to the meristem is the inva-sion zone, where rhizobia are released from infectionthreads into the host cell cytoplasm and are entrappedby plant-derived membranes in symbiosomes. Proxi-mal to the invasion zone is interzone II-III of matura-tion and the onset of nitrogen fixation. The nitrogenfixation zone constitutes the bulk of the nodule andconsists of large host cells packed with functionalbacteroids alternating with uninfected plant cells. Thecharacteristic pink color of this zone is given byleghemoglobin, a high-affinity oxygen carrier thathelps to establish the low oxygen levels required fornitrogenase activity. At a certain stage during noduledevelopment, nitrogen fixation ceases and senescenceis triggered in the proximal part of the nodule adjacentto the root (Van de Velde et al., 2006). Nodule senes-cence is a complex process that has not yet beenclarified. In our study, the expression of MtSGR wasfound to be low in most organs but was inducedduring seed maturation and nodule senescence. Be-
Figure 7. Dark incubation of alfalfa MsSGR-RNAi transgenic lines. A,Detached leaves of the wild type, empty vector control (CTRL), andMsSGR-RNAi transgenic lines in the dark. B, Living plants of the wildtype and a transgenic line (SGRi-39) in the dark. Numbers (0, 5, 10, 15,and 20) indicate days after dark treatment (DAD).
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cause SGR is associated with seed color, its increasedexpression during seed maturation is not surprising.What was unexpected was the increase in MtSGRexpression during nodule senescence. Nodule senes-cence was initiated after NO3 treatment, and theexpression level of MtSGR was induced 8-fold in M.truncatula as a result. Increased expression of SGR inM. truncatula was also associated with natural senes-cence in older nodules.
We also found that a selection of nodule senescencemarkers displayed differential expression patterns inthe sgr mutant compared with wild-type senescingnodules. This finding points to a possible role ofMtSGRin certain steps of nodule senescence. The fact thatMtSGR regulates leaf senescence and also appears to beinvolved in nodule senescence is not completely sur-prising. Previously, cDNA amplification fragmentlength polymorphism analysis identified 545 tran-scripts that were differentially expressed during M.truncatula leaf senescence (De Michele et al., 2009) and508 transcripts that were regulated duringM. truncatulanodule senescence (Van de Velde et al., 2006). While theoverlap between these two processes in terms of thegene families involved was found to be relatively low,only 7% of the leaf data set, these findings indicate theexistence of conservedmechanisms governing both leafand nodule senescence (De Michele et al., 2009).
In addition to being up-regulated during nodulesenescence, MtSGR is also expressed in all nodulezones in the wild type, suggesting that SGRmay play abroader role during nodule development and is notrestricted to senescence.
MtSGR Plays a Broad Role in the PlantSenescence Process
It is known that leaf senescence is accompanied bythe degradation of proteins, lipids, and nucleic acids(Buchanan-Wollaston et al., 2003). Our microarrayexperiments revealed nearly 1,600 genes that were
Figure 8. Measurement of chlorophyll content and Fv/Fm values in control and independentMsSGR-RNAi transgenic alfalfa linesduring dark incubation. A, Change of chlorophyll content during dark incubation. fw, Fresh weight. B, Chl a/b ratio of the samesamples as in A. C, Fv/Fm values. CTRL, Control plants. Numbers (0, 5, 10, 15, and 20) indicate days after dark treatment (DAD).Error bars indicate SE (n = 3).
Figure 9. Phenotypic appearance of control and MsSGR-RNAi trans-genic alfalfa under natural drying conditions. A, Control plants. B,SGRi-39 transgenic line. 0d, 5d, 10d, 15d, and 20d indicate days aftercutting of the materials.
Zhou et al.
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either down- or up-regulated in the sgrmutant. Basedon the GO analysis, we observed that theMtSGR genedirectly or indirectly affects the expression of a largeportion of genes involved in different metabolic pro-cesses and biosynthetic processes. These data supportthat MtSGR plays multiple roles in the senescingprocess and may explain the multiple effects on otherorgans, including anthers, seeds, and seed pods, inthe mutant. Transcript profiling was used to charac-terize a stay-green mutant (nan) of Citrus sinensis.Detailed analysis showed that the nan mutation isdistinct from sgr (Alos et al., 2008). The global profil-ing analysis of the mtsgr mutant provides valuabledata for further research in elucidating functionalmechanisms of SGR.To date, the mechanism of SGR function remains
unknown (Hortensteiner, 2009). Transcript profilingrevealed that only a small portion (2.5%) of the geneswith altered expression are associated with chloro-plast-related proteins, indicating that the role of SGRmay extend beyond chlorophyll degradation. Sinceno obvious change was observed in genes involved inthe chlorophyll degradation pathway, it is unlikelythat SGR directly affects a specific component of thechlorophyll catabolism pathway. Furthermore, thenumber of repressed genes was much smaller thanthe number of up-regulated genes in the mutant,indicating that SGR may play a preferential role ingene repression. Furthermore, MtSGR is involved innodule development and senescence, indicating thatMtSGR may be related to nitrogen remobilization. InArabidopsis and rice, SGR has been shown to localizeto the chloroplast (Park et al., 2007; Ren et al., 2007).Therefore, it is possible that the changes in transcriptprofiling of the sgr mutant are the consequences ofsecondary effects. A clearer picture can be drawn bycomparing transcript profiling data of different spe-cies after such information becomes available in otherplants.
Down-Regulation of MsSGR Improves the Forage
Quality of Alfalfa
Cultivated alfalfa or lucerne is the most importantforage legume in the United States, with approxi-mately 9.6 million ha in production and an estimatedvalue for alfalfa hay alone of US$8.1 billion (Bouton,2007). Most of the alfalfa grown in the United States isused for hay production. Color is an important char-acteristic of hay. The ideal color is one that mostclosely resembles the bright green color of an imma-ture legume crop in situ. Senescence causes leafyellowing and reduces the market value of hay. Be-cause alfalfa is a tetraploid species with a high degreeof self-incompatibility, recessive mutations can beeasily masked after crossing. To date, there has beenno report of successful conventional breeding ofalfalfa with delayed leaf senescence. In this study,silencing the MsSGR gene using the RNAi approachled to the production of stay-green transgenic alfalfa.This beneficial trait offers the opportunity to pro-duce premium alfalfa hay with a more greenishappearance.
Another important aspect of forage quality is theactual nutritive value of hay. Routine forage analysesusually include levels of crude protein, dry matterdigestibility, acid detergent fiber, neutral detergentfiber, total digestible nutrients, as well as mineralcontents (Collins and Fritz, 2003). Leaf senescencenegatively affects the nutritional quality of foragecrops. Chlorophyll degradation and the progressiveloss of protein in the chloroplasts happen duringsenescence and chloroplast degeneration (Lim et al.,2007). It has been shown that the leaf stay-greenfeature of the rice sgr mutant is associated with afailure in the destabilization of the light-harvestingchlorophyll-binding protein complexes (Park et al.,2007). Thus, delayed chlorophyll breakdown duringleaf senescence may benefit the conservation of leafprotein. This hypothesis was supported by the data ofnutritive quality analysis in MsSGR-RNAi transgenicalfalfa. Most of the transgenic lines showed a highlevel of chlorophyll retention and a simultaneoussignificant increase in crude protein content. Further-more, the analysis of chloroplast ultrastructure alsosupported that the chloroplast breakdown wasdelayed in senescent leaves of transgenic alfalfa.Because the alfalfa plants used for forage quality anal-ysis were not inoculated with rhizobia and grown insoil with full nutrition, the increase in crude proteincontent in transgenic alfalfa is not related to the nitro-gen-fixing nodules. These data suggest that the crudeprotein content is positively associated with chloro-phyll retention in transgenic alfalfa and that the in-creased chlorophyll stability has more positive impactbeyond the visual phenotype. On the other hand,down-regulation ofMsSGR did not have any obviouslynegative impact on plant phenotype and other qualitytraits, such as in vitro dry matter digestibility and totaldigestible nutrients. These results further support the
Figure 10. Crude protein content of control (CTRL) and MsSGR-RNAitransgenic alfalfa lines. Five-week-old plants were collected for anal-ysis. Error bars indicate SE (n = 3). Columns labeledwith the same lettersare not significantly different at P = 0.05.
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potential usefulness of the transgenic materials foralfalfa improvement.
In summary, we identified stay-green mutants in themodel legume M. truncatula, characterized the SGRgene, and successfully applied the knowledge to al-falfa improvement. We found that SGR not onlyplayed an important role in leaf senescence but wasalso involved in nodule senescence. Transcript profil-ing revealed that large numbers of genes were eitherup-regulated or down-regulated in the mutant. Sig-nificant improvement in forage quality was achievedin the transgenic alfalfa lines down-regulated withMsSGR. One expected outcome from the study of themodel plantMedicago truncatula is that the informationobtained would benefit alfalfa improvement (Choiet al., 2004; Zhu et al., 2005). This study illustratesthe effective use of a model system for the geneticimprovement of an important commercial crop.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Generation of theMedicago truncatula Tnt1 insertional mutant population was
described previously by Tadege et al. (2008).M. truncatula ecotype R108 was used
as the wild type. Mutant and wild-type seeds were scarified with concentrated
sulfuric acid and treated at 4�C for 5 d on filter paper. Small plantlets were
transferred to Metro-Mix 830 soil mix and grown in the greenhouse or growth
chamber under the following conditions: 24�C day/22�C night temperature, 16-
