Correction

PLANT BIOLOGYCorrection for “Overexpression of the maize Corngrass1 micro-RNA prevents flowering, improves digestibility, and increasesstarch content of switchgrass,” by George S. Chuck, ChristianTobias, Lan Sun, Florian Kraemer, Chenlin Li, Dean Dibble,Rohit Arora, Jennifer N. Bragg, John P. Vogel, Seema Singh,Blake A. Simmons, Markus Pauly, and Sarah Hake, which ap-peared in issue 42, October 18, 2011, of Proc Natl Acad Sci USA(108:17550–17555; first published October 10, 2011; 10.1073/pnas.1113971108).The authors note that the following statement should be

added to the Acknowledgments: “The work conducted by theJoint BioEnergy Institute was supported by the Office of Science,Office of Biological and Environmental Research, of the USDepartment of Energy under Contract DE-AC02-05CH11231.”

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www.pnas.org/cgi/doi/10.1073/pnas.1120302109

Overexpression of the maize Corngrass1 microRNAprevents flowering, improves digestibility, andincreases starch content of switchgrassGeorge S. Chucka,1, Christian Tobiasb, Lan Sunc, Florian Kraemerd, Chenlin Lic, Dean Dibblec, Rohit Arorac,Jennifer N. Braggb, John P. Vogelb, Seema Singhc, Blake A. Simmonsc, Markus Paulyd, and Sarah Hakea,1

aPlant Gene Expression Center/US Department of Agriculture (USDA) and University of California (UC) Berkeley, Albany, CA 94710; bUSDA-AgriculturalResearch Service Western Regional Research Center, Albany, CA 94710; cJoint BioEnergy Institute, Emeryville, CA 94608; and dEnergy BiosciencesInstitute/UC Berkeley, Berkeley, CA 94720

Contributed by Sarah Hake, September 3, 2011 (sent for review June 17, 2011)

Biofuels developed from biomass crops have the potential to sup-ply a significant portion of our transportation fuel needs. Toachieve this potential, however, it will be necessary to developimproved plant germplasm specifically tailored to serve as energycrops. Liquid transportation fuel can be created from the sugarslocked inside plant cell walls. Unfortunately, these sugars are in-herently resistant to hydrolytic release because they are containedin polysaccharides embedded in lignin. Overcoming this obstacle isa major objective toward developing sustainable bioenergy cropplants. The maize Corngrass1 (Cg1) gene encodes a microRNA thatpromotes juvenile cell wall identities and morphology. To test thehypothesis that juvenile biomass has superior qualities as a poten-tial biofuel feedstock, the Cg1 gene was transferred into severalother plants, including the bioenergy crop Panicum virgatum(switchgrass). Such plants were found to have up to 250% morestarch, resulting in higher glucose release from saccharificationassays with or without biomass pretreatment. In addition, a com-plete inhibition of flowering was observed in both greenhouseand field grown plants. These results point to the potential utilityof this approach, both for the domestication of new biofuel crops,and for the limitation of transgene flow into native plant species.

Plant biomass can be broken down to monosaccharides (sac-charification) and converted to fuels. Plant cell walls arecomposed of cellulose microfibrils embedded in a cross-linkednetwork of matrix polysaccharides and copolymerized with lig-nin. This complex structure inhibits the saccharification of cellwall polysaccharides by cell wall degrading enzymes. Further-more, byproducts of the harsh pretreatments necessary to enablesaccharification inhibit growth of microorganisms used to pro-duce biofuels. Therefore, improving saccharification efficiency isone of the major goals in developing an efficient, cost-effectivebiofuel industry (1). Because a wealth of genetic and molecularresources exists for maize, it is well suited as a model system forthe identification of genes important for biomass accumulationand saccharification in grasses (2).Plants go through a series of development stages over time in

response to a variety of stimuli, both external and internal. Eachphase displays unique morphological and physiological charac-teristics that change when the plant undergoes a transition to thenext phase. One such developmental transition is the switch fromthe juvenile to adult phase of development (3). In general, juvenileplant material is less lignified and displays differences in biomassaccumulation and character. These juvenile traits may reduce therecalcitrance of biomass to conversion into fermentable sugars. Bycontrolling the genes that regulate the juvenile to adult phasetransition in plants, it may be possible to modify or enhance thebiomass properties of a wide range of bioenergy feedstocks.The dominant maize Corngrass1 (Cg1) mutant fixes plant de-

velopment in the juvenile phase and affects both biomass accu-mulation and saccharification efficiency. Cg1 mutants increasebiomass due to continuous initiation of axillary branches (tillers)and leaves (4). The resulting biomass has reduced adult char-

acteristics and ectopic juvenile cell identities (5). In addition, Cg1mutant leaves contain decreased amounts of lignin and increasedlevels of glucose and other sugars compared with wild type (6),which could provide an improved substrate for saccharification.We cloned Cg1 and showed that it is an unusual grass-specific

tandem microRNA gene that is overexpressed in the mutant (7).This microRNA belongs to the miR156 class that is known totarget the SQUAMOSA PROMOTER BINDING LIKE (SPL)family of transcription factors (8). Overexpression of miR156causes inappropriate cleavage of its targets, demonstrating thatthe Cg1 mutant phenotype is caused by loss of function of severalSPL target genes (7). miR156 is temporally regulated, occurringat high levels during the juvenile phase and gradually dis-appearing during the adult phase in Arabidopsis and maize (7, 9).Overexpression of miR156 in Arabidopsis and rice results inphenotypes similar to Cg1 (10, 11), indicating functional con-servation of this microRNA across a wide range of plant species.SPL genes have a variety of functions, including control of

male fertility (12), copper metabolism (13), plastochron time (14,15), bract suppression (16), and flowering time (17, 18). Re-cently, it has been shown that SPL genes directly target andactivate expression of flowering regulators such as LEAFY andAPETALA1 (19), and a different microRNA, miR172 (17). Ex-pression of miR172 during the adult phase of developmentpromotes flowering by repressing floral repressors such as theSMZ and SNZ genes (20, 21). Thus, the miR156-targeted SPLgenes have a positive effect on the floral transition throughmultiple independent pathways. Consistent with these findings,the Cg1mutant in maize is slow to flower and has greatly reducednumbers of floral and inflorescence meristems (7).Because members of the miR156 targeted SPL gene family are

conserved in different plant species, it is feasible to transfer thedesired biofuel processing properties of Cg1 into any crop ofchoice simply by overexpressing the Cg1 cDNA. Here we test thishypothesis by constitutively expressing Cg1 in the monocots,Brachypodium distachyon (Brachypodium), Panicum virgatum(switchgrass), and a dicot, Arabidopsis. We show that it is pos-sible to affect biomass composition, digestibility, and floweringthrough genetic manipulation of miR156 and its targets.

ResultsCg1 Overexpression Promotes Juvenile Leaf Traits. The full-lengthmaize Cg1 cDNA was expressed behind the 35S promoter inArabidopsis or behind the maize UBIQUITIN (UBI) promoter

Author contributions: G.S.C. designed research; G.S.C., L.S., F.K., C.L., D.D., R.A., J.N.B., andJ.P.V. performed research; C.T. contributed new reagents/analytic tools; G.S.C., S.S., B.A.S.,M.P., and S.H. analyzed data; and G.S.C. and S.H. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. E-mail: georgechuck@berkeley.edu orhake@berkeley.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113971108/-/DCSupplemental.

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(22) in Brachypodium and switchgrass using Agrobacterium-me-diated transformation. Similar to the maize Cg1 mutant (Fig.1A), Arabidopsis 35S::Cg1 transformants (Fig. 1B) displayed in-creased vegetative branching and increased leaf initiation in linewith earlier reports of miR156 overexpression (10). These plantsflowered approximately 2 to 3 wk later and had morphologicallynormal flowers (Fig. 1B). UBI::Cg1 transformants in Brachypo-dium displayed a similar increase in leaf production, branching,and delayed flowering time (Fig. 1C), but also produced inflor-escences with fewer spikelets (Fig. 1C, Inset). The same constructwas put into switchgrass with similar results (Fig. 1D), except thatthe plants never flowered. In general, leaves in all speciesexpressing Cg1 were narrow and more numerous compared withwild-type adult leaves.Plastic sections of Cg1 overexpressing leaves of grass species

revealed numerous differences in cell number, morphology, andidentity (Fig. 2 A–C). In general, Cg1 overexpressing leaves inmaize, switchgrass, and Brachypodium had fewer cell layers, re-duced leaf thickness, and fewer hypodermal sclerenchyma cells(lignified support cells) (arrows) compared with wild-type adultleaves of similar age. Epidermal peels of leaves were performedon switchgrass to determine whether the transformants had juve-nile cell identity. In maize, wild-type juvenile waxes stain purplewith Toluidine Blue (TBO), whereas adult waxes stain aqua (23),and the same is true in switchgrass (Fig. 2 D and E). All leaves ofthe Cg1 overexpressors displayed juvenile waxes on the basis ofTBO staining (Fig. 2F). In addition, epidermal cells were lesscrenulated and had thinner walls compared with wild type (Fig.2F). Cg1 overexpressers in both maize and switchgrass also showedincreased juvenile biochemical properties such as an increase inthe juvenile and grass-specific, cell wall (1,3;1,4)-β-D-glucans (Fig.S1) (24). In summary, the leaves of all species that overexpressCg1 had both juvenile morphology and cell identities relative tonontransgenic plants, consistent with the maize Cg1 phenotype.

Morphological and Molecular Analysis of Ubi:Cg1 Switchgrass. Tofully understand the impact of the Cg1 mutants on the phenotypeand composition of switchgrass, 20 independent UBI::Cg1 switch-grass transformants were generated and sorted into three pheno-

typic classes (severe, moderate, and weak) (Fig. 3A) on the basisof phenotypic severity and expression levels of the transgene(Fig. S2). In general, increasing levels of Cg1 expression werecorrelated with increasing phenotypic severity. Each class dis-played increased vegetative branching, but also possessed uniquephenotypes. High transgene expressers were severely dwarfedand had thinner stems with many small needle-like leaves. Mod-erate expressers had bigger leaves, thicker stems, and were lessdwarfed. Both severe and moderate expressers produced longhorizontally growing branches that initiated several aerial shootswith roots at each node (Fig. 3B), each capable of producing newplants. The leaves of the low-expressing weak class resembledwild type, except that they were narrower and had juvenile cellidentity. In addition, the weak expressers only produced excessbranches at nodes located at soil level.The growth rate of each clone was assessed over the summer

in the field (details in Materials and Methods). Transformantsfrom the severe class with high levels of transgene expressiondisplayed very limited growth and often died, whereas the lowexpressers generally kept pace with wild-type controls (Fig. 3C).None of the transformants ever flowered, even after more than ayear in the field (two summers and one winter) or after 2 y in thegreenhouse under long day conditions. In contrast, transfor-mants overexpressing a miR156 target mimic construct, designedto inhibit miR156 activity (25), flowered normally.At the end of the growing season, all of the above ground

biomass was harvested, dried, and weighed. Postharvest, theweakly expressing class displayed better regenerative capacity,with each cut shoot forming a new tiller. In contrast, wild-typeplants only regenerated new shoots from the periphery of theplant (Fig. 3D). A count of the total number of primary branchespostharvest revealed that the weak-expressing class producedfour times more branches, whereas the moderate and severeclasses had only two to three times more, respectively (Fig. 3F).The average weight of the moderate and severe classes was sig-nificantly lower than wild type, but the weak class was not sig-nificantly lower (Fig. 3E). These results indicate that high levels

Fig. 1. Overexpression of Cg1 in different plant species. (A) Cg1 over-expression in maize (Left) compared with wild-type sibling (Right). (B) Cg1overexpression in Arabidopsis (Left) compared with wild-type Columbia(Right). (C) Cg1 overexpression in Brachypodium (Left) compared with wild-type Bd21-3 (Right). Inset shows inflorescence morphology. (D) Cg1 over-expression in switchgrass (Left) compared with wild-type Alamo (Right). Allplants are the same age and grown under the same conditions.

Fig. 2. Histological analysis of Cg1 overexpressors. (A) Plastic sections of ju-venile, adult, and Cg1 leaves of maize. (B) Plastic sections of juvenile, adult,and UBI::Cg1 leaves of Brachypodium. (C) Plastic sections of juvenile, adult, andUBI::Cg1 leaves of switchgrass. (D) Epidermal peel of juvenile switchgrass leaf.(E) Epidermal peel of adult switchgrass leaf. (F) Epidermal peel of UBI::Cg1switchgrass leaf. All plastic sections and epidermal peels were stained withToluidine Blue. Arrows point to lignified hypodermal sclerenchyma cells.

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of Cg1 expression are detrimental to plant growth and biomassaccumulation, whereas weak expression can be tolerated.To confirm that overexpression of Cg1 in switchgrass reduces

SPL gene expression, qRT-PCR was performed using oligoscorresponding to several switchgrass SPL homologs (Fig. 3G).As expected, the expression levels of four miR156-targeted SPLgenes were down by several orders of magnitude in all trans-formants, the most dramatic found in the moderate and severeclasses. Because SPL genes are known activators of AP1 MADSbox genes necessary for specifying floral meristem identity (19,26), the expression of three switchgrass homologs of AP1 wasalso assayed (Fig. 3H). Two of these genes, FL779848 andFL813474, were down-regulated in all transformants, whereasthe third one, FL799727, showed no significant change. Assum-ing that FL779848 and FL813474 function similarly to AP1, theloss of these genes could explain the lack of flowering in thetransformants. Because SPL genes are also known activators ofmiR172, it was predicted that targets of miR172 would showa converse expression pattern to the SPLs, and therefore be up-regulated in the transformants. This prediction was confirmed inthe weakly expressing class with a putative switchgrass homologof the maize glossy15 gene, FL805250, which functions to repressadult leaf cell characteristics (27) (Fig. 3I). The ectopic expres-sion of this gene may help explain the extended juvenile leaf cellcharacteristics of the Cg1 transformants in switchgrass, maize,and Brachypodium.

Biochemical Properties of Ubi:Cg1 Plants. Composition analysis ofdried UBI::Cg1 switchgrass material was carried out to assess itsamenability to biofuel production. Total lignin content of driedwhole plants was measured using acetyl bromide assays (28) andshowed modest reduction in all three classes of transformants

(Fig. S3A), compared with both wild type and miR156 targetmimic transformants used as a negative control. This finding isconsistent with earlier lignin measurements of Cg1 leaves inmaize (6). Cellulose levels and the amount of cellulose relative tolignin (Fig. S3 B and C) showed a modest increase in all threeclasses. In addition, two-step sulfuric acid hydrolysis followed byhigh-performance anion exchange chromatography (HPAEC)was used to measure monosaccharide levels (Fig. S3D) andrevealed that each class of transformants had slightly higherglucan levels and lower xylan levels. These biochemical differ-ences in biomass properties, although slight and significant onlyin the moderate class, indicated that these plants might be moreeasily saccharified.

Ubi:Cg1 Plants Store Starch in Their Stem. Pretreatment of plantbiomass is often used to make it more accessible to enzymebreakdown and fermentation. Saccharification assays using dilutealkali pretreatment demonstrated significantly higher glucoserelease in the severe switchgrass transformants (Fig. 4A) com-pared with maize, Brachypodium, and Arabidopsis Cg1 plants(Fig. S4). By contrast, saccharification assays on Cg1 switchgrassstems and leaves performed using acid pretreatment showed nosignificant difference in total glucose release over time in thetransformants compared with wild type (Fig. S5). These differ-ences suggest that some fermentable biomass components mightbe made accessible only by specific pretreatment regimes. Be-cause the switchgrass plants never flower, we hypothesized thatthis difference might be due to alterations in stored carbohy-drates, because many nonflowering Arabidopsis mutants displayhigher starch levels (29). Indeed, potassium iodide-stained handsections of field grown leaves and stems demonstrated a strikingdifference in starch levels in stem segments of Cg1 switchgrass

Fig. 3. Morphological and molecular analysis of UBI::Cg1switchgrass plants. (A) Phenotypes of three classes of Cg1switchgrass transformants. (B) Close up of a severe trans-formant showing ectopic shoots at each node. (C) Com-parison of weakly expressing transformant (Left) and wildtype (Right) in the field. Wild-type plant is flowering. (D)Same plants in C, 3 wk postharvest. The transformant dis-plays better recovery. (E) Comparison of first year post-harvest dry weights of above-ground biomass of wild typeand three classes of transformants. (F) Comparison of tillernumbers of wild type and three classes of transformants.For data in E and F error bars show 1 SE. Sample sizes: WT,n = 4; weak, n = 4; moderate, n = 6; and severe, n = 7.Asterisks indicate significant differences with wild type(Materials and Methods). (G) qPCR analysis of wild typeand three classes of transformants using primers corre-sponding to four different miR156-targeted SPL genes. (H)qPCR analysis using primers corresponding to three dif-ferent AP1 MADS box genes. (I) qPCR analysis using primerscorresponding to a switchgrass glossy15 homolog.

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plants compared with wild-type controls (Fig. 4B). In post-flowering wild-type switchgrass, starch is normally found in thelowermost nodes, whereas in Cg1 lines it is present in every node(Figs. S6 and S7). High levels of starch accumulation were notobserved in maize, Brachypodium, and Arabidopsis Cg1 linesusing the same potassium iodide assay.Starch levels were measured directly from the stems of field

grown transformants harvested before dawn. The weakly expres-sing transformants had >250% more starch in the stem, whereasthe moderately expressing transformants had up to 189% more(Fig. 4C). The severe class had low starch presumably due to thefact that the tissue was a mixture of stem and leaf. Because theweak class of transformants appeared to grow at near normalrates compared with wild type in the field, they were chosen forfurther in depth analysis.Additional saccharification assays of the weakly expressing

transformants were performed without biomass pretreatment.Because the weak-expressing class had more starch, α-amylase andamyloglucosidase were either added to the saccharification en-zyme mix to digest it (method II) or not (method I). Using methodI, only a modest increase in glucose release was seen (Fig. 4D).However, three to four times more glucose was released fromstems using method II after 24 h (Fig. 4D). The increase in glucoserelease from these transformants also displayed improved kineticswhen measured over 72 h (Fig. 4 E and F). In fact, the amount ofglucose release over time from weakly expressing stems usingmethod II without pretreatment is similar to the amount derivedfrom the same stems pretreated with dilute acid (Fig. S5). Thisfinding indicates that Cg1 overexpression may either reduce ornegate the pretreatment requirement for saccharification. Thus,weak Cg1 overexpression appears to transform the stem intoa starch-containing storage organ, allowing for significantly higherglucose release in saccharification assays without pretreatmentand without severely compromising plant growth.

DiscussionThe maize Cg1 gene was overexpressed in several plants to fixthem in the juvenile phase of development and determine itsimpact in the context of biofuel production. Cg1 is a uniquetandem miR156 microRNA gene that, to date, has been identi-fied only in grass species (7) and may have been under purifyingselection during domestication (30). The unique structure of thisgene may play a role in transcript stability because the full-lengthCg1 transcript can be easily detected on RNA blots (7) in con-trast to many miR156 precursors. In addition, overexpression ofCg1 causes a slightly different suite of phenotypes compared withsimple miR156 overexpression in Arabidopsis (10). BecausemiR156 genes are highly conserved and have been identified inall sequenced plant genomes to date, it is feasible to fix any plantof choice in the juvenile phase simply by overexpressing the Cg1cDNA. Cg1 overexpression in maize, Arabidopsis, Brachypodium,and switchgrass produced plants with extra branches and leavesthat possess juvenile morphology, cell identity, and biochemicalproperties, confirming that high levels of miR156 are sufficient toinduce juvenile development. Previous analysis of juvenile bio-mass in maize showed that it possessed decreased lignin andincreased levels of certain sugars (6), making it a superior sub-strate for fermentation.The Departments of Energy and Agriculture have identified

switchgrass as a potential bioenergy crop that may reduce ourreliance on fossil fuels (31). Ethanol produced from switchgrassbiomass is projected to produce more than 500% more renew-able energy than the energy consumed on the basis of lifecycleanalysis and would emit 94% fewer greenhouse gases than gas-oline (32). Switchgrass is a warm season C4 perennial grass withno vernalization requirement and can flower under long daysfollowed by short days (33, 34). It can flourish on marginalcropland, does not compete with food crops, and requires min-imal growth inputs. Taking into consideration the ecologicalimpacts such as soil conservation, improvement in water quality,wildlife habitat restoration, and reduction in carbon emissions

Fig. 4. Biofuel properties and digestibility of UBI::Cg1 switchgrass. (A) Saccharification assay using di-lute base pretreatment of field grown leaves of wildtype and three classes of transformants. Asterisksindicate significant differences with wild type. (B)Potassium iodide staining of upper nodes of fieldgrown stems of Cg1 (Left) and wild type (Right). (C)Starch measurements of field grown stems of wildtype and two independent transformants of eachclass. The severe class contained leaf bases. (D) Sac-charification assay of stems and leaves of wild typeand two independent weak transformants with-out biomass pretreatment after 24 h of digestion.Method I was done with Accelerase enzyme mixture,and method II was done with the same mixture plusα-amylase and amyloglucosidase. (E) Saccharificationassay over 72 h of stems of two field grown wild-typeplants and two weakly expressing transformants us-ing method I without pretreatment. (F) Saccharifi-cation assay over 72 h of stems of two field grownwild-type plants and two weakly expressing trans-formants using method II without pretreatment. Alltransformants in D–F were assayed in duplicate.

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(35), it is clear that switchgrass has numerous potential envi-ronmental benefits in addition to its economic ones. Usingtransgenic technology to improve the biofuel properties of thisplant is a feasible way to quickly establish it as a viable bioenergycrop at a commercial scale in the United States and the world.Here, we tested the hypothesis that juvenile biomass of switch-grass could represent an enhanced feedstock for thebiofuel industry.We show that Cg1 switchgrass tissue is easier to digest and

releases more glucose in saccharification assays of specific tissuesusing either alkaline pretreatment (Fig. 4A) or no pretreatment(Fig. 4 D–F). The ability to release glucose in the absence ofpretreatment is due to the presence of starch in stems that is onlyfully released through addition of starch degrading enzymes tothe saccharification mix. Thus, juvenile biomass has uniqueproperties, both histologically and biochemically, which togethermake it an attractive target for modification to improve biofuelproduction. It is clear, however, that high levels of ubiquitousCg1 expression have detrimental effects on overall plant growth(Fig. 3E). To address these problems, constructs driving Cg1behind weakly expressing tissue-specific promoters are pending.An unexpected consequence of Cg1 overexpression in switch-

grass was the absence of flowering in both the greenhouse and thefield, even after more than 2 y of growth. Lack of flowering wasnot seen in any other plants overexpressing miR156, includingmaize, Arabidopsis, and rice (4, 10, 11) and could reflect a novelrole for SPL genes in controlling flowering in switchgrass. Severalflowering time genes have been identified and analyzed in mon-ocots, including CONSTANS, EARLY HEADING DATE,HEADING DATE 3A, and INDETERMINATE (36). None ofthese genes, however, showed any significant expression differ-ence in the switchgrass transformants compared with wild type.The SPL transcription factors regulated by miR156 are knownregulators of AP1 MADS box transcription factors (19). In fact,the first SPL gene was cloned on the basis of its ability to bind thepromoter of the AP1 ortholog in Antirrhinum (26). The AP1 geneis a regulator of floral meristem identity and is the last step in theArabidopsis flowering pathway (37). Two of three switchgrassAP1 homologs were greatly reduced in expression in the Cg1transformants, and a reasonable hypothesis is that several ofthese AP1 genes are required for floral initiation in switchgrass.Alternatively, the switchgrass plants may not flower as a conse-quence of increased expression from AP2 gene targets ofmiR172, which function as floral repressors (21). Overexpressionof miR156 is known to repress miR172 in grasses (7), causingtargets of miR172 to be up-regulated. In support of this obser-vation, a switchgrass homolog of the miR172 AP2 target geneglossy15 is up-regulated in the Cg1 weakly expressing lines,making it likely that other related miR172 floral repressors areup-regulated as well.Lack of flowering in UBI::Cg1 switchgrass has important

implications, not only for biomass quality, but for prevention oftransgene escape into native plant populations. A major im-pediment to using new transgenic crop plants is the need forcontainment of either transgenic seed or pollen. Spread oftransgenes from pollen and seed dispersal has the potential to bea major ecological problem for growers using transgenic crops(38). The fact that Cg1 switchgrass does not flower presentsan effective solution to this problem. Moreover, in grasses suchas wheat, the flowering signal initiates reallocation of carbonresources from stem tissue to reproductive tissue, including seeds(39). By restricting flowering, this carbon stays in vegetative tis-sue, which should allow for increased carbohydrate levels asa basis for fermentation. Finally, in some annual plants floweringcan act as a signal that promotes senescence and causes loss ofvegetative carbon (40). Carbon loss from senescence is reducedin Cg1 transformants, and in fact, many transformants are ef-fectively immortalized. The fact that Cg1 stems contain greaterthan twofold more starch supports the idea that flowering andstarch degradation are interconnected. It is possible that starch

destined for use during floral development is left unused in theCg1 transformants, and thus accumulates in stems.Cg1 switchgrass aerial stems store starch and initiate both

roots and shoots, giving them both the appearance and functionof a rhizome. It is possible that the lower juvenile nodes of allgrasses have rhizome potential, and that overexpression ofmiR156 extends this potential to the upper regions of the plant.Although additional studies are needed to determine whetherthe presence of excess starch may make plants more susceptibleto predation, we show that having extra starch may minimize theneed for extensive pretreatment of the biomass. Starch is mucheasier to digest compared with cellulose and can simply be re-leased through saccharification with amylase enzymes, which aresignificantly cheaper that cell wall degrading enzymes. Circum-venting the pretreatment requirement also results in significantenergy savings, because this process requires very high temper-atures and the use of caustic chemicals. In addition, unlike otherbioenergy crop plants such as sugarcane, dried starchy biomasscan be stored long term and is easy to transport. Ultimately, thisinformation can be readily transferable to other less geneticallytractable species such as Miscanthus and sorghum, and may im-prove a variety of different plants for use as biofuel crops.

Materials and MethodsVector Construction and Transformation. The maize full-length Cg1 cDNA wascloned into the maize Ubiquitin cassette vector pUbi-BASK-Nos cassette, andthen blunt-end ligated into the Agro binary vector pWBVec8. This binaryvector was transformed into Alamo switchgrass (41) and into BrachypodiumBd21-3 (42). The same cDNA fragment was cloned into pENTR (Invitrogen),recombined into Agrobacterium binary pKGW, containing the 35S promoterNOS 3′ cassette, and then transformed into Arabidopsis Columbia plants byfloral dipping.

Histology. For plastic sections, plants were fixed overnight in FAA, dehydratedin an ethanol series, and embedded in JB-4 plastic per the manufacturer’sinstructions. Two-micrometer sections were made and stained for 30 s inTBO and then photographed using darkfield optics. For epidermal peels,hole punches of juvenile, adult, and Cg1-overexpressing leaves were made,fixed in 1% formaldehyde in PBS, washed several times with water, and thenincubated with 0.1% Pectinolyase overnight (43). The next day, the epi-dermis was peeled off, dipped into TBO, washed, and photographed usingbrightfield optics.

Field Trials and Branch Counts. Twenty independent UBI::Cg1-overexpressingswitchgrass transformants and four wild-type plants were grown in 4 × 4inch pots for 1–2 mo in the greenhouse, cut back, and then transplanted intothe field in Berkeley, California in early June 2010. Total above-groundbiomass from 17 of the surviving lines was harvested in late October 2010and dried for 3 wk at 37 °C in a corn dryer before analysis. Tiller numberswere determined after harvest by counting stem stubs. To date, all Cg1transformants have still not flowered either in the field or in the green-house, whereas target mimic negative control lines flowered after 6 mo.

To calculate significance, dry weight and primary branch number datawere analyzed using a two-tailed, Student’s t test with unequal variance.*P < 0.0001.

qPCR Analysis. To quantify transcript levels, qPCR was done on a Bio-Rad CFX-96 real-time PCR machine. Five hundred nanograms of poly(A)+ purified RNAwas used for each cDNA synthesis reaction. Approximately 5–10 ng of cDNAwas included into each PCR with single color real-time detection using SybrGreen. All PCR reactions were done with three biological replicates of eachsample and two technical replicates and normalized against the switchgrassactin gene as a reference using the delta Ct method. All primer sequencesare available in SI Materials and Methods.

Analysis of Starch. Freeze dried plant material from two biological replicatesof each transformant class and two wild-type plants was ground for 1 min toa fine powder in a Kleco ball mill 8200 (Kleco). Alcohol insoluble residue wasprepared by washing the samples with ethanol (70% vol/vol), chloroform/methanol (1:1 vol/vol), and acetone (once, thrice, and once, respectively). Thesamples were dried in a speed vac.

Approximately 100 mg of alcohol insoluble residue (AIR) was dispensedinto 15 mL conical polypropylene tubes in duplicates. The samples were

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wetted with 200 μL ethanol (80% vol/vol). Immediately, 3 mL of thermo-stable α-amylase (100 U/mL in 100 mM sodium acetate buffer, pH 5.0) wasadded and the tubes incubated for 12 min at 100 °C in a Techne DB-3Bheater with intermittent rigorous mixing after 4, 8, and 12 min. The sampleswere cooled on ice to room temperature and 100 μL of amyloglucosidase(3,300 U/mL) was added. After rigorous mixing, the samples were incubatedfor 30 min at 50 °C. The volume of the samples was adjusted to 10 mL withdistilled water and the samples were centrifuged for 10 min at 3,000 rpm.An aliquot of 1 mL of supernatant was transferred to a 2-mL screw-cappedmicrocentrifuge tube and the glucose concentration was measured on theYSI 2700 Select Biochemistry Analyzer (YSI Life Sciences). Two technicalreplicates were performed for each sample.

Starch staining was done on razor blade hand sections of two biologicalreplicates of field grown stems boiled in 95% ethanol, and then dipped inLugol’s potassium iodine solution, and then rinsed in water and mountedin glycerol.

Enzymatic Saccharification of Dilute Base Pretreated Biomass. Field grownleaves were freeze dried and treated with dilute base, ground to a finepowder, and treated with Accellerase 1500 enzyme mix (Gencor). Each assaywas done with ∼2 mg of material in 2-mL screw-cap tubes in 1 mL of 50 mMcitrate buffer (pH. 4.5) including sodium azide to a final concentration of0.01 mg/mL, at 50 °C under constant (mild) agitation for 20 h. A total of 2 μLof enzyme was used per sample. The Megazyme D-Glucose kit was used tomeasure the glucose content following the manufacturer’s procedures on sixtechnical replicates on single plants from each transformant class.

Dilute Acid Pretreatment. Switchgrass samples from two biological replicatesfor each transformant class including two wild-type plants were presoaked in

pressurized glass tubes at room temperature in 1.2% (wt/wt) sulfuric acid at3% (wt/wt) total solid loading for at least 4 h. The glass tubes were thenheated at 160 °C for 30 min. The acidic slurry was filtered through Whatmanfilter paper with a Buchner funnel. The recovered solids were washed withdeionized water until the pH of the washed water reached 5.0.

Enzymatic Saccharification of Untreated and Dilute Acid Pretreated Biomass.Batch enzymatic saccharification of pretreated and untreated biomass samplesfrom two biological replicates of each class were carried out at 50 °C and 150rpm in a reciprocating shaker. Two enzyme mixtures were used. One mixturewas Accellerase 1500 (method I) and the other mixture contained Accellerase1500 plus α-amylase and amyloglucosidase (method II). Each assay was donewith ∼10 mg of material in 5 mL of 50 mM citrate buffer (pH. 4.5), includingsodium azide, to a final concentration of 0.01mg/mL, 10 μL of Accellerase 1500plus 10 μL of Megazyme thermostable α-amylase, and 10 μL amyloglucosidase(only with method II) was added to each sample. Sampling was performed at0, 0.5, 1, 2, 3, 24, 48, and 72 h. The Megazyme D-Glucose kit was used tomeasure the glucose content following the manufacturer’s procedures. Twotechnical replicates were performed for each sample.

Additional methods and references can be found in SI Materialsand Methods.

ACKNOWLEDGMENTS. We thank Thant Niang for his help on the project,including the plastic sections and branch counts. Thanks go to David Hantzfor greenhouse management and China Lunde for reviewing the manuscriptand help with statistics. This work was supported by Department of Energy(DOE) Grant DE-A102-08ER15962 and Binational Agricultural Research andDevelopment Grant IS-4249-09 (to S.H. and G.S.C.) and by DOE Grant DE-SC0004822 (to M.P. and S.H.).

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