A Rice PECTATE LYASE-LIKE Gene Is Required for PlantGrowth and Leaf Senescence1[OPEN]

Yujia Leng2, Yaolong Yang2, Deyong Ren2, Lichao Huang, Liping Dai, Yuqiong Wang, Long Chen,Zhengjun Tu, Yihong Gao, Xueyong Li, Li Zhu, Jiang Hu, Guangheng Zhang, Zhenyu Gao,Longbiao Guo, Zhaosheng Kong, Yongjun Lin, Qian Qian*, and Dali Zeng*

State Key Lab for Rice Biology, China National Rice Research Institute, Hangzhou 310006, China (Yu.L., Y.Y.,D.R., L.H., L.D., Y.W., L.C., Z.T., Y.G., L.Z., J.H., G.Z., Z.G., L.G., Q.Q., D.Z.), National Key Laboratory ofCrop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University,Wuhan 430070, China (Yu.L., L.D., L.C., Yo.L.), National Key Facility for Crop Gene Resources and GeneticImprovement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China(X.L.), and Institute of Microbiology, Chinese Academy of Sciences, State Key Laboratory of Plant Genomics,Beijing 100101, China (Z.K.) State Key Lab for Rice Biology, China National Rice Research Institute, Hangzhou310006, China (Yu.L., Y.Y., D.R., L.H., L.D., Y.W., L.C., Z.T., Y.G., L.Z., J.H., G.Z., Z.G., L.G., Q.Q., D.Z.),National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research,Huazhong Agricultural University, Wuhan 430070, China (Yu.L., L.D., L.C., Yo.L.), National Key Facility forCrop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of AgriculturalSciences, Beijing 100081, China (X.L.), and Institute of Microbiology, Chinese Academy of Sciences, State KeyLaboratory of Plant Genomics, Beijing 100101, China (Z.K.)

ORCID IDs: 0000-0001-7797-5162 (L.H.); 0000-0002-8955-0345 (X.L.); 0000-0003-4949-8164 (G.Z.); 0000-0003-3788-7883 (Z.K.);0000-0002-0349-4937 (Q.Q.); 0000-0003-2349-8633 (D.Z.).

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified amutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histologicalanalysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that thedecline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, andTUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 genewas cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain.DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues butpredominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymaticactivity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. Inaddition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leafsenescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATELYASE-LIKE genes.

The development of higher plants is accompanied bythe complex processes of cell division and cell expansionand finally the emergence of many specialized organs,tissues, and cells. As part of cell development in plants,cell walls perform essential functions, such as providingtensile strength, shape, and protection, as well as helpingthe cell to maintain its internal pressure (Cosgrove, 2005).In growing plants, the cell wall is both resistant to thehigh turgor pressure that drives growth and alsoflexible enough to yield and expand selectively in re-sponse to that pressure (Cosgrove, 2005; Xiao et al., 2014).This process involves a series of molecular mechanisms,including disruption of the intermolecular adhesion,polymer lysis, religation and rearrangements, and/orcleavage of polymer glycosyl linkages by enzymes(McQueen-Mason and Cosgrove, 1995; Van Sandt et al.,2007; Anderson et al., 2010; Park and Cosgrove, 2012).

1 This research was supported by the National Natural ScienceFoundation of China (Grant No. 91435105, 3161143006, 91535205),National Key Basic Research Program (grant no. 2013CBA014), andthe “Science and technology innovation project” of the Chinese Acad-emy of Agriculture Sciences.

2 These authors contributed equally to the article.* Address correspondence to dalizeng@126.com or qianqian188@

hotmail.com.The author responsible for the 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:Dali Zeng (dalizeng@126.com).

Yu.L., Y.Y., and D.R. performed research; L.H., L.D., Y.W., L.C.,Z.T., Y.G., L.Z., J.H., G.Z., Z.G., L.G., Yo.L., X.L., and Z.K. analyzedthe data; Yu.L. wrote the article; Yu.L., D.Z., and Q.Q. designed theresearch; D.Z. revised the article.

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Pectins are major components of the plant primarycell wall and middle lamella and have several functions,including involvement in maintaining plant growth anddevelopment, promoting cell-to-cell adhesion, providingstructural support in soft tissues, defense responses, andinfluencingwall porosity and thickness, etc (Ridley et al.,2001; Iwai et al., 2002; Ogawa et al., 2009; Wolf et al.,2009; Hongo et al., 2012). Pectins may be the mostcomplex polysaccharide family in the livingworld, beingcomposed of as many as 17 different monosaccharidesand having more than 20 different linkages (Bonninet al., 2014). According to the current understanding ofpectins at the structure/function level, there are twoconceptual models of structural modification duringplant growth (Atmodjo et al., 2013). First, the largenumber of linkages and structural motifs allow the do-mains of pectins to interact indirectly and/or via cova-lent bonds (Dick-Pérez et al., 2011; Tan et al., 2013).Second, pectins can depend on reversible calcium-mediated cross linking between stretches of demethy-lated homogalacturonan (HG) to coordinate cell wallnetworks (Vincken et al., 2003; Xiao et al., 2014). HG issecreted in a highly methylesterified form and selec-tively demethylesterified by pectin methylesterases(PME; Hongo et al., 2012). The demethylesterified HGcan either form a rigid gel by Ca2+-pectate cross-linkedcomplexes, or become more susceptible to cleavage bytwo classes of pectin-degrading enzyme, namely pec-tin/pectate lyase (PL/PEL; EC 4.2.2.10; EC 4.2.2.2) andpolygalacturonase (PG; EC 3.2.1.15) (Wolf et al., 2009;Hongo et al., 2012). Hence, the methylesterificationstatus of HG can regulate cellular growth and cellshape, and affect plant growth and development (Wolfet al., 2009; Peaucelle et al., 2011).

PELs, a family of endo-acting depolymerizing en-zymes, are responsible for the a-1,4-glycosidic linkagesin demethylesterified HG by b-elimination and pro-duces 4,5-unsaturated oligogalacturonides (OGs) attheir nonreducing ends (Palusa et al., 2007). PELs havebeen extensively studied in plant pathogenic bacteriasuch as Erwinia chrysanthemi, which causes soft-rot dis-eases (Barras et al., 1994). In plants, multiple functionshave been identified for PEL, and there is some sug-gestion that it is involved in pollen, anthers, pistils, anddeveloping tracheary elements (Wing et al., 1990; Rogerset al., 1992;Wu et al., 1996; Kulikauskas andMcCormick,1997; Domingo et al., 1998; Milioni et al., 2001), fruitsoftening and ripening (Dominguez-Puigjaner et al.,1997;Medina-Escobar et al., 1997;Nunan et al., 2001; Puaet al., 2001), lateral root emergence (Laskowski et al.,2006), cotton fiber elongation (Wang et al., 2010), leafsenescence (Wu et al., 2013), and susceptibility to plantpathogens (Vogel et al., 2002). It has also been shown tobe expressed in a wide range of tissues (Palusa et al.,2007; Sun and van Nocker, 2010). In addition, recentresearch has revealed that the overexpression of aspenPtxtPL1-27 can increase the solubility of wood matrixpolysaccharides (Biswal et al., 2014). Themodification ofpectins may be important in the field of biotechnologyfor improving woody biomass (Biswal et al., 2014).

The large family of PECTATE LYASE-LIKE (PLL)genes all exhibit redundant or unique functions in plantevolution, and this diversity may enhance plasticity inadaptation to changing environments (Sun and vanNocker, 2010). In rice (Oryza sativa), genome predictionhas suggested that there are 14 PLL genes, but only oneof these (ospse1) has been analyzed genetically (Wuet al., 2013). In this study, we report on the isolation andcharacterization of a recessive mutant, dwarf and early-senescence leaf1 (del1), in rice. The mutation of DEL1 ledto a phenotype involving abnormal plant growth andleaf senescence. DEL1 encodes a PEL precursor and is amember of a multigene family in rice. The loss of func-tion of DEL1 decreased total PEL activity, increased thedegree of methylesterified HG, and perturbed cell wallcomposition and structure, resulting in a reduced num-ber of cells and triggering reactive oxygen species (ROS)activity. Expression profiling indicated the genes in-volved in cell wall function and senescence are altered inexpression in the del1 plants. Our findings suggest thatDEL1 is a critical gene for plant growth and leaf senes-cence in rice.

RESULTS

del1 Exhibits Significant Size Reduction in the Whole Plant

The del1 mutant was isolated from an ethylmethanesulfonate-mutagenized japonica cultivar, Nip-ponbare. del1 exhibited dwarfism at the seedling stage (5 dafter germination), and its main root length was clearlyreduced, which reached 47.8% of that in the wild type(Fig. 1, A and C). Meanwhile, the number of lateral rootsin del1 decreased significantly to just 44.7% of that in thewild type (Fig. 1, B and D). The plant height of del1wasalso shorter than that of the wild type throughout thegrowthperiod, being only about 36.8%of that of thewildtype at the mature stage (Fig. 1, E and F; SupplementalFigs. S1 and S2), and the internode length, thickness, anddiameter of del1were significantly reduced (SupplementalFig. S3). Compared with the wild type, the tiller numberand panicle lengthwere also clearly reduced in del1 plants(Fig. 1, E, G, H, andK).Moreover, grain development alsochanged significantly, with grain size and grain weightbeing diminished in del1 plants (Fig. 1, I, J, and L;Supplemental Table S1). Besides these phenotypicfindings, the heading date of del1 was also notablyretarded, along with decreases in leaf length and leafwidth (Supplemental Table S1). These phenotypesclearly suggest that plant size was significantly lowerin del1 plants.

Cell Number Is Reduced in del1 Plants

The size of a plant organ is determined by its cell sizeand number of cells, which are related to cell expansionand cell division, respectively (Krizek, 2009). To deter-mine the causes of organ size reduction in del1 plants,we conducted microscopic observation on the second

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culms and compared the findings between the wildtype and del1 using paraffinized sections. Cross sectionsof culms revealed that the size of sclerenchyma cells indel1 was less than that in the wild type (Fig. 2, A–D).Statistical analysis showed that the cell number of del1wasonly 88.56% of that in the wild type (Fig. 2E). Moreover,the number of layers of sclerenchyma cells in del1 wasreduced by two (Fig. 2, C, D, and F). Longitudinal sectionsof culms in del1 displayed a significant change in cell size(Fig. 2, G andH). The cell length in del1was 59.6% longer,and the cell width was 20.8% narrower than that in thewild type (Fig. 2, I and J). Further investigation of the totalnumber of parenchyma cells showed that del1 had only19% of the number in the wild type (Fig. 2K).

Cell Cycle Progression Is Delayed in del1 Plants

To determine whether the decline in cell number indel1 was affected by the cell cycle, we further investi-gated the cell cycle progression by flow cytometry. Theresults of the suspension cell lines of del1 revealed a

significant increase in the number of cells in the G1phase and decreases in the S and G2/M phases of thecell cycle, implying that the cell cycle was delayed at theG1 phase (Fig. 3, A–C).We also analyzed the expressionof cell-cycle-related genes in wild-type and del1 plantsusing real-time reverse transcription (RT)-PCR. Theexpression levels of genes related to the G1 phase of thecell cycle, CDKA1, CAK1, CAK1A, MCM5, and CYCT1,were down-regulated by ;10% to 45%, while little dif-ferencewas observed for genes related to theG2phase ofthe cell cycle in del1 mutants (Fig. 3D). Therefore, weconclude that the mutation of DEL1 delays cell cycleprogression at the G1 phase.

del1 Displays Early Leaf Senescence

del1 also exhibited a phenotype of early senescence,which became increasingly apparent as the plants de-veloped (Fig. 4, A–D). The mutant leaf apex and leafmargin exhibited a faint yellow color from 5 d aftergermination (Fig. 4A). With the development of plants,

Figure 1. Comparison of phenotypebetween wild-type and del1 plants. A,Wild-type (Nipponbare, left) and del1plants (right) 5 d after sowing. Scalebar = 2 cm. B, Root length of wild-type(left) and del1 plants (right) 5 d aftersowing. Scale bar = 1 cm. C and D,Statistical analysis of root length andlateral root number between wild-typeand del1 plants. Twenty plants were mea-sured. Error bars indicate SD; **P , 0.01(Student’s t test). E,Wild-type (left) anddel1plants (right) atmaturity. Scale bar=10cm.F and G, Statistical analysis of plant heightand tiller number between wild-type anddel1 plants. Twenty plants were measured.Error bars indicate SD; **P , 0.01 (Stu-dent’s t test). H, Phenotype of paniclebetween wild-type (left) and del1 (right)plants. Scale bar = 5 cm. I, Floret withthe lemma removed, wild-type (left)and del1 (right). Scale bar = 0.5 cm. J,Mature seed and brown rice of wildtype (left) and del1 (right). Scale bar =0.5 cm. K and L, Statistical analysis ofpanicle length and thousand grainweight between wild-type and del1plants. Twenty panicles were mea-sured. Error bars indicate SD; **P, 0.01(Student’s t test).

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the young leaves were pale white and then turnedgreen upon maturation, while the old leaves exhibitedwithering and cracking (Fig. 4, B–D). The chlorophyllcontent in del1 plants was significantly lower, being only44.0% of that in the wild type (Fig. 4E). In addition, trans-mission electron microscopy (TEM) revealed the presenceof well-developed mesophyll cells and membrane-intactchloroplasts in fully developed wild-type leaves, whereasthe disordered arrangement of grana thylakoid and thedegradation of chloroplasts was observed in del1 plants(Fig. 4, F and G). Moreover, the photosynthetic rate indel1 plants was only 58.5% of that in the wild type (Fig.4H). All these results indicate that leaf senescence oc-curred in del1 plants.

Leaf senescence is usually accompanied by a change ofexpression of many genes, including those for transcrip-tion factors (Wang et al., 2015). To confirm senescence inthe del1 plants, the expression levels of senescence-associated genes (SAGs) and related transcriptionfactors (Osl2, OsH36, SGR, OsNAC2, OsWRKY23,and OsWRKY72) were determined by real-time RT-PCR.The expression levels of Osl2, OsH36, SGR, OsNAC2,

OsWRKY23, and OsWRKY72 mRNAs were 2.0 to 8.0times higher than in wild-type leaves (Fig. 4I). Theup-regulated expression patterns of SAGs and tran-scription factors further support the notion that earlyleaf senescence occurred in del1 plants.

ROS Accumulation and Programmed Cell Death AreEnhanced in del1 Plants

The accumulation of ROS can lead to leaf senescence(Khanna-Chopra, 2012). Therefore, we performed nitroblue tetrazolium (NBT) staining and 3,39-diaminobenzidine(DAB) staining tests to detect O2

2 and H2O2 accumulation,respectively. Extensive NBT staining was observed indel1 plants, whereas the staining was minimal in wild-type leaves (Fig. 5A). A brown color was seen for theDAB staining, correlating with the area of leaf senes-cence, but there was no sign of this in wild-type leaves(Fig. 5B). These results revealed that ROS accumulatedin del1 plants. During senescence, plant cell membranedamage can lead to a change in electrolyte leakage(Blum and Ebercon, 1981). In del1 plants, the electrolyte

Figure 2. Histological characteriza-tion of culms in wild-type and del1plants. A to D, Cross sections of in-ternode II of wild type (A) and del1(B). Scale bar = 500 mm. C, Magnifi-cation of A; D, magnification of B;white rectangle shows a magnifica-tion of the sclerenchyma cell layer.Scale bar = 50 mm. E and F, Statisticalanalysis of cell number and scleren-chyma cell layer number betweenwild-type and del1 plants, means 6SD of five independent replicates. Gand H, Longitudinal sections of in-ternode II of wild type (G) and del1(H). Scale bar = 50 mm. I and J, Sta-tistical analysis of the cell length andcell width between wild-type anddel1 plants, mean 6 SD of 30 cells.**P , 0.01 (Student’s t test). K,Number of parenchyma cells (PCs) forinternode II of wild-type and del1plants, mean6 SD of five independentreplicates.

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leakagewas 45.8% higher than that in the wild type (Fig.5C), suggesting that del1 lost more membrane integrityduring development than the wild type.Plant senescence can lead to the synthesis of anti-

oxidative enzymes to remove ROS (Miller et al., 2010).Therefore, we quantitatively determined the activity ofthese enzymes. The results indicated that the activities ofsuperoxide dismutase (SOD) and peroxidase (POD) in-creased in del1 plants, at 100.3 and 110.4 U/mg freshweight in del1 leaves, which were much higher than thelevels of 62.2 and102.3U/mg freshweight in thewild-typeleaves, respectively (Fig. 5, D and E). In the process of leafsenescence, ROS-scavenging systems may play an

important role in ROS detoxification (Tan et al., 2014).ROS-scavenging-related genes (alternative oxidases[AOXs], ascorbate peroxidase [APX], SOD, and cata-lase [CAT]) were up-regulated during leaf senescence(Tan et al., 2014). As expected, the expression levels ofROS-scavenging-related genes (AOX1a, AOX1b, APX1,APX2, SODB, SODA1, catA, and catB) were 2.5 to 13.6times higher than in the wild type (Fig. 5F).

Leaf senescence is the final stage of leaf development,and it is considered to be a type of programmed cell death(PCD; Nooden and Leopold, 1978). Trypan blue stainingrevealed that the leaf apex and leaf margin of del1 werecoloredblue, but the counterpartwild type remainedgreen(Fig. 5G), suggesting that some of the cells in the leaf apexand leaf margin of the del1 plant were dead. One basicfeature of PCD is the condensation of nuclear chromatin,which is caused by endonucleolytic degradation of nuclearDNA (Simeonova et al., 2000). We accordingly used theTUNEL assay to determine whether the del1 plants in-duced PCD. A few of the nuclei in the wild type wereTUNEL positive; in contrast, most nuclei in the del1 leafsectionswereTUNELpositive (Fig. 5,H–K), indicating thatDNA degradation was widespread in the del1 leaves.

Map-Based Cloning of DEL1

To determine the molecular basis for the del1 phe-notypes, a map-based cloning approach was employedto isolate the corresponding gene. The mapping popu-lation was generated by crossing DEL1 with TaichungNative 1, a wild-type indica variety with DNA poly-morphism with japonica. All the F1 plants exhibited anormal phenotype matching that of the wild type. Inthe F2 segregating populations, normal and mutantphenotypes showed a typical segregation ratio of 3:1(Supplemental Table S2). This finding suggests that del1is controlled by a single recessive nuclear gene.

Thirty F2 plantswith the del1phenotypewere used forprimarymapping, andDEL1was found to be located onthe long arm of chromosome 10 between markers M1and M14. Furthermore, by using 1081 homozygousmutant plants, DEL1 was further narrowed down toan ;45-kb region with molecular markers as shown inSupplemental Table S5. This 45-kb region is in the BACAC025905 and contains eight putative open readingframes (ORFs), as annotated by the MSU Rice GenomeAnnotation Project (Fig. 6A). Upon sequencing analysisof these eightORFs, one base pairmutationwas found atposition 1940 of ORF LOC_Os10g31910. This changefrom G to T causes a substitution at the 365th amino acidresidue fromTrp to Leu (Fig. 6B). By examining this base insix other rice varieties, we found that they all exhibited thewild-type genotype (Supplemental Fig. S4).

To confirm that LOC_Os10g31910 is responsible for thedel1 phenotype, a 7843-bp wild-type DEL1 DNA fragmentcontaining the promoter region and the entire ORF wasintroduced into del1 plants by Agrobacterium tumefaciens-mediated transformation. Analysis of independenttransgenic plants by PCR using primers flanking themutation site, togetherwith further sequencing, revealed a

Figure 3. Cell cycle analysis of wild-type and del1 plants. A and B, Flowkaryotype histogram of wild-type (A) and del1 (B) leaves. C, Quantificationof the DNA profiles of wild-type and del1 plants. D, Relative expressionlevels of cell cycle-related genes inwild-type and del1 plants,mean6 SD ofthree independent replicates. *P , 0.05, **P , 0.01 (Student’s t test).

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double peak (G and T) at themutation site, confirming thepresence of normal DEL1 gene sequence (Fig. 6C). Thetransgenic plants rescued the phenotypes of del1, whereasin all of those transformed by pCAMBIA1300, there was afailure to rescue the mutant phenotype, confirming thatdisruption of the DEL1 gene was responsible for the del1mutant phenotype (Fig. 6, D–I). In addition, RNAi trans-genic plants exhibited early senescence leaves and a sig-nificantly reduced size (Supplemental Fig. S5, A–E). Theexpression of DEL1 was significantly decreased in RNAiplants (Supplemental Fig. S5F). Therefore, we concludethat LOC_Os10g31910 is the DEL1 gene.

DEL1 Encodes a Pectate Lyase Precursor

Sequence analysis indicated thatDEL1 cDNA is 1476 bpin length and encodes a protein of 491 amino acid residues(Fig. 7A). A search using SignalP and Pfam revealed thatthe DEL1 protein contains a 28-amino acid signal peptideand one predicted PelC domain (Fig. 7A). Multiple

sequence alignment analyses revealed thatDEL1 containsall the conserved residues of PELs known to be involvedin Ca2+ binding, substrate binding, and catalysis and hasstronger similarity with plant PelC than Erwinia chrys-anthemi (Supplemental Fig. S6).

An unrooted phylogenetic tree was built among PELmembers of rice, Arabidopsis (Arabidopsis thaliana), andseven known plant PELs using the neighbor-joiningmethod, which revealed that PELs were divided intofive major clades, with clade I being further dividedinto four subclades (Fig. 7B). DEL1 belongs to Ic and isin the same clade as PMR6 (a powdery mildew sus-ceptibility) of Arabidopsis (Vogel et al., 2002).

DEL1 Was Highly Expressed in Elongating Tissues

To analyze the spatial and developmental expressionof the DEL1 genes, we isolated RNA from differenttissues, including young roots, young sheaths, youngleaves,mature roots,mature sheaths,mature leaves, culms,

Figure 4. Leaf phenotype and identifica-tion of leaf senescence in DEL1. A to D,Leaf phenotype fromyoung (A), tillering (Band C), and heading (D) stages. Scalebars = 1, 5, 5, and 1 cm in A, B, C, andD, respectively. E, Statistical analysis ofchlorophyll content between wild-typeand del1 plants, mean 6 SD of five inde-pendent replicates. **P , 0.01 (Student’st test). F and G, Transmission electronmicroscopy analysis of senescence leavesof wild-type (F) and del1 plants (G). Scalebar = 0.5 mm. H, Statistical analysis ofphotosynthesis rate between wild-typeand del1 plants, mean 6 SD of five inde-pendent replicates. **P, 0.01 (Student’st test). I, Relative expression levels of se-nescence-related genes and transcriptionfactors in wild-type and del1 plants,mean 6 SD of three independent repli-cates. *P , 0.05, **P , 0.01 (Student’st test).

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panicles, and spikelets, and determined the transcriptlevels of the DEL1 genes by real-time RT-PCR. The resultsrevealed that DEL1 was ubiquitously expressed in all thetissues examined at the young andmature stages, andhighlevels of DEL1 expression were observed in elongatingtissues, such as culms and roots (Fig. 8A).In addition, to analyze the spatial expression pattern

of DEL1, we expressed the GUS gene under the control

of the native promoter of the DEL1 gene. Six indepen-dent DEL1:GUS transgenic lines were analyzed, and allof them exhibited similar results. The GUS activity wasexpressed in all of the tissues and was consistent withthe qRT-PCR data (Fig. 8, B–J).

Mutation of DEL1 Decreased Total PEL Activity andAltered Cell Wall Composition and Structure

Since PelC is the only domain identified in DEL1, itmay be critical for its function. Therefore, we deter-mined the total PEL activity. As expected, PEL enzymeactivity was significantly decreased in del1 plants. Es-pecially in culms, the PEL activity was only 32.0% ofthat in the wild type (Fig. 9A). This result is consistentwith the real-time RT-PCR (Fig. 8A) and the ProDEL1:GUS expression results (Fig. 8, B–J).

The morphological phenotypes suggest that the cellwall structure in the mutant plants may also be altered.We therefore analyzed the structure of wild-type anddel1 plants by TEM. The results revealed that the wallthicknesses of bundle sheath fiber cells in del1 culmswere altered (Fig. 9, D–G). Specifically, the thicknesses ofthemiddle lamella and secondary cellwallwere reducedby 51.6% and 40.6%, respectively, and that of the pri-mary cell wall was 15.6% higher than those of the wildtype (Fig. 9, H–J). Similarly in roots, the thicknesses(sclerenchyma cell) of the middle lamella and secondarycell wall were reduced by 18.5% and 41.4%, respectively,and that of the primary cell wall was 12.7% higher thanthose of the wild type (Supplemental Fig. S7).

To determine whether changes in the cell wallstructure affected the wall composition, we analyzedthe cellulose, hemicellulose, and pectin contents andcompared them between wild-type and del1 culms. Thecellulose content of del1 was reduced by ;20%, whilethe contents of hemicellulose 1 (HC 1), hemicellulose2 (HC 2), and pectin were increased by 58.3, 27.3, and117.2%, respectively (Fig. 9B). In addition, the levels ofseven neutral monosaccharides were also determinedand compared between the wild-type and del1 plants.All the neutral monosaccharides in del1 exhibited sig-nificant increases, except for Xyl (Fig. 9C). These resultssuggest that the DEL1 mutation causes complex com-positional and structural alterations in cell walls.

Mutation of DEL1 Increased the Degree ofHG Methylesterification

Given that PEL can catalyze the a-1,4 glycosidiclinkages of demethylated HG by b-elimination, we usedimmunohistochemical techniques to discern in situ as-pects of cell wall microstructures and locate polymersprecisely. The second culms from wild-type and del1plants were probed using JIM5, LM18, and LM19 anti-bodies, which are used to recognize partially demethy-lesterified and unesterified HG; JIM7 antibody, whichlabels moderately high extent of methylesterified HG;and 2F4 antibody, which binds to HG with degrees ofmethylesterification up to 40% (Verhertbruggen et al., 2009;

Figure 5. ROS accumulation and enhancement of PCD in wild-type anddel1 leaves. A and B, NBT and DAB staining of leaves between the wild-type (left) and del1 plants (right). C to E, Statistical analysis of electrolyteleakage (C), SOD activity (D), and POD activity (E) in leaves betweenwild-type and del1 plants, mean6 SD of five independent replicates. **P, 0.01(Student’s t test). F, Relative expression levels of ROS detoxification-relatedgenes in wild-type and del1 plants, mean 6 SD of three independent rep-licates. **P , 0.01 (Student’s t test). G, Trypan blue staining of leaves inwild-type (left) and del1 plants (right). H to K, TUNEL assay of leaves. DAPIstaining of wild-type (H) and del1 plants (J). Positive results of wild-type (I)and del1 plants (K). Scale bar = 50 mm.

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Heldet al., 2011).As shown inFigure 10, immunolabelingofculm sectionswith JIM7, LM18, LM19, and 2F4 indicated anapparent increase in the recognition of HG epitopes, whileJIM5 exhibited a slight increase, suggesting that the degreeof methylesterified HG was higher in del1 plants.

Activation of Cell Wall- and Senescence-Related GenesRevealed by Transcriptome Analysis

To further unravel the function ofDEL1 in rice, 10-d-oldwild-type and del1 plants were used for transcriptome

analysis. A total of 491 differentially expressed genes(DEGs) were found with a P value, 0.05 (SupplementalTable S3). Among these genes, 332were up-regulated and159 were down-regulated in del1 plants (SupplementalTable S3). Clusters of orthologous groups of proteinsfunctional catalog showed that in DEGs were assigned to39.2% and 50.9% of the up- and down-regulated genes,respectively (Supplemental Table S3). Most of the genesup-regulated in del1 were involved in secondary metab-olite biosynthesis, transport, and catabolism, carbohy-drate transport and metabolism, and lipid transport and

Figure 6. Map-based cloning and iden-tification of DEL1. A, Fine mapping ofDEL1. The del1 locus was mapped toa 45-kb region on chromosome 10. B,Schematic diagram of DEL1. Black rect-angles represent exons. Black invertedtriangle represents mutant site. C, Se-quencing analysis of theDEL1 transcriptsin T0 transgenic lines.D and E, Phenotypeof the complementation transgenic line:Wild type (left), complementation trans-genic line (middle), and empty vectorcontrol (right). Scale bars = 10 cm and4 cm, respectively. F, Expression levels ofDEL1 detected by qRT-PCR in wild-typeand transgenic plants, mean6 SD of threeindependent replicates. G to I, Statisticalanalysis of plant height (G), tiller number(H), and flag leaf length (I) in wild-typeand transgenic plants, mean 6 SD of10 independent replicates.

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metabolism (Supplemental Table S3). The majority of thedown-regulated genes were associated with translation,ribosomal structure and biogenesis, secondary metabo-lites biosynthesis, transport, and catabolism, and cellwall/membrane/envelope biogenesis (SupplementalTable S3). Of note, a set of the DEGs associated withcell wall and senescence were significantly altered indel1 plants, such as the PME gene (OsPME1), b-expansingene (OsEXPB2), peroxidase precursor gene (OsPOX1),protochlorophyllide oxidoreductase B gene (OsPORA),etc. (Kim et al., 2012; Sakuraba et al., 2013; Zou et al.,2015; Fang et al., 2016; Supplemental Table S4). Theseresults suggested that the mutation ofDEL1might affectthe expression of cell wall- and senescence-related genesin an indirect manner.

DISCUSSION

PEL is an endogenous pectin-degrading enzyme thatis capable of cleaving a-1,4-glycosidic linkages indemethylated pectin by b-elimination (Palusa et al.,2007). It is a ubiquitous enzyme in higher plants and isencoded by at least 26, 22, and 14 genes in Arabidopsis,poplar (Populus trichocarpa), and rice, respectively (Palusaet al., 2007). Although severalPLL genes are considered toplay potentially diverse physiological roles in plants, suchas being expressed in anthers and pollen, and being in-volved in fruit softening and ripening, pathogen defense,and tissue/plant growth and development, their molec-ular mechanism in monocots remains largely unknown.Here, we identify DEL1 as a PEL precursor in modelmonocot rice using a forward genetic approach; it con-tains a PelC domain and represents a multigene family in

Figure 7. Prediction of the primary sequence and phylogeneticanalysis of DEL1. A, The deduced amino acid sequence of DEL1.Numbers on the left refer to the positions of amino acid residues.The signal peptide is indicated with an underline; the PelC domainis shown by a dotted line; the conserved residues involved in Ca2+

binding (red background), disulfide bonds (orange background),catalysis (blue background), and substrate binding (purple back-ground). B, Phylogenetic tree of PEL in Arabidopsis, rice, and otherplants. The numbers at each node represent the bootstrap support(percentage), and scale bar is an indicator of genetic distance basedon branch length.

Figure 8. Expression analysis ofDEL1. A, Transcription level ofDEL1 invarious organs, mean 6 SD of three independent replicates. YR, Youngroot; YL, young leaf; YS, young sheath; MR, mature root; C, culm; ML,mature leaf; MS, mature sheath; P, panicle; S, spikelet. B to J, GUSanalysis of DEL1 expression: B and C, Four and seven days after ger-mination of the young plant, scale bar = 1 cm; D, root, scale bar =500mm; E, lateral root, scale bar = 250mm; F,mature sheath, scale bar =1 cm; G, mature leaf, scale bar = 1 cm; H, culm, scale bar = 1 cm; I,spikelet, scale bar = 1 cm; J, lemma and palea were removed in I, scalebar = 500 mm.

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rice.DEL1 is crucial for plant growth and leaf senescence.The loss of function of DEL1 reduced the total PEL en-zymatic activity, increased the degree of methylesterifiedHG, and perturbed the cell wall structure and composi-tion, resulting in retardation of the cell cycle and en-hancement of ROS activity. These results reveal a dualmolecular function of PLL genes in rice.

DEL1 Impacts Plant Growth by Regulating CellCycle Progression

Cell division/expansion is a fundamental, dynamiccellular process driving plant growth and develop-ment, and it enables the plant and various organs todevelop to suitable sizes (Duan et al., 2012). The orderlydevelopment process involves many genes and path-ways that affect plant organ size by altering cell num-ber, cell size, or both (Krizek, 2009). Although recent

studies have uncovered some key regulators and genesthat affect plant organ size, the intrinsic mechanismsresponsible for organ size variation are yet to be fullyunderstood (Krizek, 2009; Duan et al., 2012). In thisstudy, we identified a PLL gene,DEL1, that appeared toplay an important role in the control of organ size inrice. In del1 plants, various organs were dramaticallyreduced in size, including in terms of root length, leafsize, plant height, grain size, and panicle and internodelength (Fig. 1; Supplemental Table 1). Inside the organs,paraffinized sections of internodes revealed that cellnumber was lower in the del1 plants (Fig. 2), suggestingthat cell division is inhibited in the mutant.

Cell division requires a range of complicated pro-cesses that must be strictly executed in a spatially andtemporally controlled manner (Dewitte and Murray,2003). The inhibition of cell division could alter cell cycleprogression and affect plant development (Dewitte and

Figure 9. The levels of PEL activity and cell wall composition and structure in wild-type and del1 plants. A, Analysis of PELactivity in wild-type and del1 plants, mean6 SD of five independent replicates. **P, 0.01 (Student’s t test). B, Comparison of cellwall composition between wild-type and del1 plants, mean6 SD of five independent replicates. **P, 0.01 (Student’s t test). HC1, Hemicellulose 1; HC 2l hemicellulose 2. C, Neutral monosaccharide composition betweenwild-type and del1 plants, mean6SE of five independent replicates, **P , 0.01 (Student’s t test). D and E, Transmission electron microscopy micrographs of thebundle sheath fiber cells of wild-type (D) and del1 (E) plants, scale bar = 1 mm. F, Magnification in D, and G. magnification in E,scale bar = 0.2mm.ml, Middle lamella; pw, primary cell wall; sw, secondary cell wall; pm, plasmamembrane; c, cytoplasm. H toJ, Statistical analysis of the middle lamella (H), primary cell wall (I), and secondary cell wall (J) thicknesses of bundle sheath fibercells between the wild-type and del1 plants, mean 6 SD of 30 cells, **P , 0.01 (Student’s t test).

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Murray, 2003). Our study demonstrates that the cellcycle in the G1 phase was significantly retarded in del1plants (Fig. 3, A–C). Recent evidence has indicated that avariety of cell cycle regulators play roles as targets cou-pling cell proliferation with development, and theirproper functioning is crucial for patterning (Ramirez-Parra et al., 2005). Consistent with the flow cytometricresults, the expression levels of genes regulating the G1phase of the cell cycle were down-regulated in del1, andno significant difference was observed in the G2 phase(Fig. 3D). These results strongly suggest that DEL1 con-trols plant growth by regulating cell cycle progression.

DEL1 Contributes to Early Leaf Senescence throughROS Accumulation

Leaf senescence is a complex process that involvesmany highly organizedmolecular and cellular changes,

such as the disintegration of chloroplasts; down-regulation of photosynthesis; degradation of nucleicacids, proteins, and lipids; and recycling of nutrients (Limet al., 2007). This process is controlled by both internal andexternal factors. To date, although many SAGs and/ortranscription factors have been identified, the complexmechanisms responsible for leaf senescence in rice remainpoorly understood. Recently, Wu et al. (2013) identifiedthe novel leaf senescence gene OsPSE1, which encodes aPEL. Mutation of OsPSE1 displays a premature senes-cence phenotype. In our study, we identified a PLL gene,DEL1, which also appears to play a general role in leafsenescence. TEM observation, physiological analysis, andTUNEL assays showed that leaf senescence was inducedin del1 plants, resulting in the withering and cracking ofleaves (Fig. 4). Moreover, the up-regulated expressionlevels of SAGs and transcription factors also demonstrateleaf senescence in del1 plants (Fig. 4I).

Figure 10. Immunohistochemical localization of HG in culm sections of wild-type and del1 plants. A to T, Immunolocalization ofHG of wild-type (A and C) and del1 plants (B andD) with JIM7, wild-type (E andG) and del1 plants (F andH) with JIM5, wild-type(I and K) and del1 plants (J and L) with LM18, wild-type (M and O) and del1 plants (N and P) with LM19, and wild-type (Q and S)and del1 plants (R and T) with 2F4. Scale bar = 50 mm.

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ROS are byproducts of various metabolic processessuch as photosynthesis and respiration in chloroplasts,mitochondria, and peroxisomes (Apel and Hirt, 2004).They can cause oxidative damage to thylakoid mem-branes and other cellular components and assumeseveral important roles in leaf senescence (Wang et al.,2015). In the present study, staining by NBT and DABindicated the accumulation of O2

2 and H2O2 in del1plants (Fig. 5, A and B), and there was also an elevatedlevel of electrolyte leakage, which is a marker of cellmembrane damage (Fig. 5C). Meanwhile, the activitiesof SOD and POD were increased in del1 plants (Fig. 5,D and E). These results indicate that leaf senescence indel1 plants is triggered by the accumulation of ROS.Under normal physiological conditions, cells controlROS levels by balancing the generation of ROS withtheir elimination by the ROS scavenging system, butthe overproduction of ROS can trigger retrogradesignaling from chloroplasts to the nucleus, resulting inalteration of the expression of nuclear-encoded genes(Tan et al., 2014). Consistent with this, the expressionof ROS-scavenging genes was significantly up-regulated(Fig. 5F). In addition, transcriptome analysis also revealedthat a set of the DEGs associated with senescence wassignificantly altered in del1 plants (SupplementalTable S4).

Taking these findings together, it is clear that ROSaccumulation-mediated chloroplast membrane break-age and chloroplast degradation play an important rolein leaf senescence in the del1 plants.

Possible Molecular Mechanism of DEL1 Responsible forthe Mutant Phenotypes

PLL genes have been shown to exhibit a broad rangeof functions in plants, which depend on the degrada-tion of demethylesterified HG and alteration of cellwall composition and structure (Yadav et al., 2009;Biswal et al., 2014). In the process of fruit ripening, thecell wall needs to loosen the xyloglucan-cellulose net-work and pectin solubilization, this processes increasingthe access of degradative enzymes to their substrates(Brummell, 2006). For pathogen defense, the accumu-lation of pectin increases hydrogen bonding in theextracellular matrix, resulting in decreased nutrientavailability to the pathogen (Vogel et al., 2002). Thedecrease of de-esterified pectin in cotton fiber canpromote its elongation (Wang et al., 2010). Our studyalso demonstrates that the mutant phenotype of del1depends on the alteration of cell wall composition andstructure. Despite our failure to purify the fusionDEL1protein via prokaryotic expression in Escherichia colior transient expression in Nicotiana benthamiana (datanot shown), the mutant phenotype indicated thatDEL1 protein plays an important role in rice growthand leaf senescence.

Many cell wall-related mutants display abnormalgrowth andmorphogenesis (Pien et al., 2001). It has beenspeculated that cell wall biogenesis andmodification are

tightly associated with cell growth via key genes at theinterface of morphogenesis, the cell cycle, and cell wallbiogenesis (Somerville et al., 2004; Zhang et al., 2010).In rice, the Brittle Culm 12 gene, encoding a kinesin-4protein, has been implicated in cell cycle progression,cellulose microfibril deposition, and wall composition(Zhang et al., 2010). In our study, the del1 mutantexhibited cell wall abnormalities and the retardation ofcell cycle progression,which indicates thatDEL1 is at theinterface between the cell cycle and cell wall biogenesis.

The dynamic interactions of plant cells depending onthe status of pectin in the cell wall form an importantregulatory mechanism of growth and development(Wolf et al., 2012). The degree of HG methylester-ification has a large effect on the physical properties ofthe cell wall (Held et al., 2011). A high degree of HGmethylesterification impedes the formation of calcium-mediated cross linking complexes, which is thought topromote cellular expansion by increasing wall flexibil-ity (Wolf et al., 2009). Our findings revealed that thedegree of methylesterified HG was increased, as de-termined by immunohistochemical assay, which indi-cates that the cell wall loosened and expanded in thedel1 plants (Fig. 10). Consistent with this, the cell lengthwas significantly increased and the expression levels ofexpansion were all dramatically up-regulated in thedel1 plants (Fig. 2, G and H; Supplemental Fig. S8).These results indicate that the function of DEL1 affectsnot only cell number, but also cell expansion.

In addition, our findings also revealed an early leafsenescence phenotype in comparison to that in wild-type plants. This initially appears paradoxical, but itcould potentially be explained by the alterations of cellwall structure. Pectin OGs are oligomers of a-1,4-linkedgalacturonosyl residues and are released by PEL- andPG-mediated breakdown of HG (Côté et al., 1998).

Figure 11. A schematic model of DEL1 function in rice. Homo-galacturonan is secreted in a highly methylesterified form and selec-tively demethylesterified by PME. The demethylesterified HG might becleaved by DEL1 and other PELs or PGs. The alternative of the cell wallregulated the cell cycle/expansion and ROS, enabling normal ricegrowth and leaf senescence process.

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Oligogalacturonides can produce ROS signal moleculesand trigger many abiotic stress responses in plants(Ferrari et al., 2013). In our study, mutation in DEL1significantly increased ROS content. We speculate thatDEL1 may participate in the OG-mediated ROS path-way that affects leaf senescence.We hypothesize a conceptual model that by tuning

HG methylesterification, cell wall component andstructure might act as downstream components of theregulatory networks that control cell cycle and expan-sion, as well as ROS, enabling normal growth and leafsenescence in rice (Fig. 11).

MATERIALS AND METHODS

Plant Materials

The rice (Oryza sativa) del1mutant was isolated from ethyl methanesulfonate-treated japonica cultivar Nipponbare, as described previously (Guo et al., 2006).An F2 mapping population was generated from a cross between del1 and anindica cultivar, Taichung Native 1 (TN1). All plants were cultivated in paddies inHangzhou (HZ, 119°549 E, 30°049 N) and Hainan (HN, 110°009 E, 18°319 N),during rice-growing seasons.

Paraffin Sectioning and TEM Analysis

For paraffin sectioning, the samples were fixed in 50% ethanol, 0.9 M glacialacetic acid, and 3.7% formaldehyde overnight at 4°C, then dehydrated with agraded series of ethanol, infiltrated with xylene, and embedded in paraffin(Sigma-Aldrich). The specimens were sectioned (8 mm thickness) with a LeicaRM2245, transferred onto glass slides with poly-L-lysine-coated, deparaffinizedwith xylene series, and dehydrated through an ethanol series (Ren et al., 2016).The sample section was conducted using a Nikon Eclipse 90i microscope.

For TEM, the samples were fixed in 2.5% glutaraldehyde in PBS for at least4 h and washed in PBS three times. Then, they were postfixed with 1% (w/v)OsO4 for 2 h after extensive washing in PBS, dehydrated with a graded ethanolseries, and infiltrated with Suprr Kit (Sigma-Aldrich). The specimens were thensectioned (70 nm ultrathin) with a Leica EM UC7 ultratome, and the sectionswere stained by uranyl acetate and alkaline lead citrate for 10 min before beingobserved by TEM with a Hitachi model H-7650.

Flow Cytometric Analysis

Cell cycle analysis was performed in accordance with the method describedby Galbraith et al. (1983). In brief, suspension cells were cut using a razor bladeand placed in ice-cold Galbraith’s buffer (45 mM MgCl2, 30 mM sodium citrate,20 mM MOPS, and 0.1% Triton X-100, pH 7.0). The homogenates were thenpassed through a 20-mm mesh to remove cellular debris. After staining with49,6-diamino-phenylindole (DAPI; 2 mg mL21), the nuclei were analyzed usingFACSAria II flow cytometer (BD Biosciences). A total of 30,000 events wererecorded, and three independent flow cytometric experiments were carried outand analyzed using the ModFit LT (Verity Software House) and FlowJo V10software (Tree Star).

Chlorophyll Content and Net Photosynthesis Rate

A total 0.2 g of samples from wild-type and del1 leaves were extracted with80% acetone. The chlorophyll content was determined according to the methodpreviously described (Yang et al., 2016). Net photosynthesis rate was measuredusing a Walz GFS-3000 (Eichenring). Parameter settings were accordance withthe manufacturer’s instructions.

Histochemical Staining and ROS-ScavengingEnzyme Assays

DAB and NBT staining was used to detect the accumulation of ROS, aspreviously described (Blum and Ebercon, 1981). Trypan blue staining was

performed as described previously (Koch and Slusarenko, 1990). Electrolyteleakage was determined in accordance with a previous study (Zhou and Guo,2009). Assays of the activities of SOD and POD were conducted in accordancewith previously described methods (Wang et al., 2013).

TUNEL Assays

The TUNEL assays were performed as described previously (Huang et al.,2007). The leaves were fixed in 4% paraformaldehyde in 0.1 M PBS containing0.1% Tween 20 and Triton X-100 at 4°C overnight and embedded in paraplasts.The sections of leaves (8 mm thickness) were treated using a Fluorescein In SituCell DeathDetection Kit (Roche). The green fluorescence of fluorescein and bluefluorescence of DAPI was analyzed using a Carl Zeiss LSM 710 laser-scanningconfocal microscope (Gottingen).

Gene Cloning and Complementation

For genetic analysis, 1081 F2 mutant plants were used for fine mappingof the DEL1 locus. The molecular markers of polymorphism are listed inSupplemental Table S5. The markers were designed using Primer 5 software(Primer-E). Gene prediction and sequence analysis were performed using thepublicly available rice databases, including Gramene (http://www.gramene.org) and the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml). For the complementation construct, a 7,843-bp genomicDNA fragment containing the entire DEL1 coding region, a 1,998-bp upstreamsequence, and an 841-bp downstream region was amplified using KOD FX(Toyobo) from Nipponbare genomic DNA and inserted into the binary vectorpCAMBIA 1300. The recombinant binary vector was introduced into Agro-bacterium tumefaciens strain EHA105 by electroporation and transformed intothe del1 mutant callus as described previously (Hiei et al., 1994). For the RNAiconstruct, a 336-bp fragment was amplified by PCR from Nipponbare cDNAand inserted into the vector pTCK303. The recombinant binary vector wastransformed into the Nipponbare callus. The primer sequences used in thisstudy are listed in Supplemental Table S5.

Phylogenetic Analysis and Protein Domain Identification

Annotated proteins of DEL1 domains were identified in the Pfam database(http://pfam.xfam.org/). The signal peptide was predicted using SignalPversion 4.1 (Petersen et al., 2011). Homologous protein sequences of DEL1were identified using the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/). Amino acid sequence alignments were conducted using ClustalX 2.1(http://www.clustal.org/) and DNAMAN version 5.0 (Lynnon Biosoft). Aphylogenetic tree was constructed using MEGA4 software (http://www.megasoftware.net/).

Gene Expression Analysis

Total RNA from roots, culms, sheaths, leaves and panicles at young andmature stages was extracted using the RNeasy plant mini kit (Qiagen). RNAreverse transcription was performed using ReverTra Ace real-time PCR-RT kitwith gDNA remover (Toyobo). After synthesis, the cDNA reaction was dilutedfive times in TE buffer, and 1 mLwas used for real-time RT-PCR using the SYBRGreen PCR Master Mix kit (Applied Biosystems) and gene-specific primers(Supplemental Table S5) in ABI7900 (Applied Biosystems). The tissue-levelexpression pattern of DEL1 was analyzed using the GUS reporter gene. Thepromoter of DEL1 (2,038 bp upstream of ATG) was amplified from the genomeDNA of Nipponbare and inserted into the binary vector pCAMBIA1305in-framewith the GUS reporter gene. The binary vector was introduced into theNipponbare callus to generate transgenic plants. The positive transgenic plantswere used to analyze the GUS reporter gene. Different tissues of T1 plants wereincubated at 37°C in 0.1 M X-Gluc in buffer [0.1 M Na2HPO4-NaH2P4, pH 7.0,10% Triton X-100, 0.5 M EDTA-Na, and 50 mM K3Fe(CN)6] for;12 h to examineGUS activity. After being cleaning in 70% ethanol, the tissues were photo-graphed under a light microscope.

Enzyme Activity Assays

PEL enzyme activity assays were routinely performed using a modifiedversion of the method of Collmer et al. (1988). A total of 0.5 g of sample wassuspended in 1.5 mL of extraction buffer (20 mM sodium phosphate, pH 7.0,

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20 mM Cys/HCl, 1% polyvinylpyrrolidone, MW 360,000, and 1 mM phenyl-methylsulfonyl fluoride) and centrifuged at 10,000g for 30min. The supernatantwas collected as the crude extract for enzyme assays.

The enzyme activity system consisted of 0.3%polygalacturonic acid in 0.07 M

Tris-HCl buffer (pH 8.0), 0.1 M CaCl2, crude extract protein, and sterile water.The enzyme reactionwas incubated at 37°C for 30min, and then stopped by theaddition of 9% ZnSO4$7H2O and 0.5 M NaOH. The control tubes received theenzyme after the addition of ZnSO4$7H2O and NaOH. One unit of PEL activitywas defined as the amount of enzyme that produces 1 mM of 4,5-unsaturatedproduct in 1 min under the assay conditions. The activity of PEL is expressed inunits per mg of protein. Soluble protein concentrations were determined inaccordance with Bradford method (Bradford, 1976), using bovine serum albu-min as a standard.

Immunofluorescence Microscopy

The culms of wild-type and del1 plant at the same development stage werefixed and embedded in glycol methacrylate. Two-micrometer-thick sectionswere cut with a Leica RM2265. Immunolabeling was performed in accordancewith the methodology of Willats et al. (2001). The primary antibodies JIM5,JIM7, LM18, LM19, and 2F4 were used at a 1:10 dilution in PBS containing 5%(w/v) fat-free milk powder (5%M/PBS) and the secondary antibody (goat anti-rat IgG coupled with fluorescein isothiocyanate) were used at dilution of 100-fold in 5% M/PBS. Sections incubated without primary antibody were used ascontrols to assess the autofluorescence of the samples. The labeled sections wereobserved using Carl Zeiss LSM 710 confocal microscope.

Compositional Analysis of Neutral Glycosyl Residues andCellulose Content

Alcohol-insoluble residues of culms were prepared as described by Zhanget al. (2012). In brief, destarched alcohol-insoluble residues samples were hy-drolyzed in 2 M trifluoroacetic acid at 121°C for 90 min. The samples werecentrifuged to collect the supernatants, air-dried, and then dissolved with a 1 M

ammonium hydroxide buffer containing NaBH4. The alditol acetate derivativeswere analyzed using an Agilent 7890 GC system equipped with a 5975C MSD(gas chromatography-mass spectrometry; Song et al., 2013). To measure thecrystalline cellulose content, the pellets remaining after trifluoroacetic acidtreatment were hydrolyzed with Updegraff reagent (Updegraff, 1969). Thesamples were subsequently centrifuged and the supernatant removed. Thepellets were then treated with 72% sulfuric acid. The cellulose content wasquantified via an anthrone assay, and three replicates were included.

Cell Wall Extraction and Measurement ofPolysaccharide Content

Cell wall materials and fractionation components were analyzed in accor-dance with themethods of Zhong and Lauchli (1993). The samples were groundin liquid nitrogen to a fine power and suspended in 75% ethanol for 25min in anice-coldwater bath. The sampleswere then centrifuged at 12,000 rpm for 15minand removed the supernatant. Next, the pellet was suspended andwashedwithprecooled acetone, followed by methanol:chloroform and then with methanol.The remaining pellet as a crude cell wall fraction was freeze-dried and stored at4°C for future use.

The cell wall extractswere fractionated into three parts: pectin,HC 1, andHC2. First, the pectin fraction was extracted twice using 0.5% ammonium oxalatebuffer containing 0.1% NaBH4 (pH 4) in a boiling water bath for 1 h. Next, thepellets were subjected to triple extractions with 4% KOH containing 0.1%NaBH4 at room temperature for 8 h each, followed by a similar extraction with24% KOH containing 0.1% NaBH4. The HC 1 and HC 2 fractions were referredto as hemicellulose material (Yang et al., 2008). The uronic acid content in thepectin fraction was assayed in accordance with the method of Blumenkrantzand Asboe-Hansen (1973) using GalUA (GalA) as a standard.

Transcriptome Sequencing and Data Analysis

The RNA samples from 10-d-old wild-type and del1 plants were used fortranscriptome sequencing, and each sample was pooled for total RNA isolationwith three biological replicates. Total RNA was extracted using TRIzol reagent(Invitrogen Life Technologies). Transcriptome sequencing was performed inBiomarker Biotechnology Corporation using the Illumina system HiSeq2500

(Illumina) according to the standard procedure. Transcriptome assembly wasperformed according to the protocol described previously (Yu et al., 2013).Differentially expressed genes were defined by using IDEG6 (Romualdi et al.,2003), at a significance level of P , 0.05. Functional annotation analysis ofDEGs in wild-type and del1 plants was performed by DAVID Web tools(Huang et al., 2009).

Accession Numbers

Sequence data from this article can be found in the EMBL/GenBank datalibraries under the following accession numbers: DEL1 (LOC_Os10g31910),PMR6 (At3g54920), NJJS25 (AF339024), PtxtPL1-27 (EU379971), ZePel(Y09541), MaPel (AAF19195), GhPel (ADB90478), CryjI (BAA05542), SGR(LOC_Os09g36200), OsNAC2 (LOC_Os01g66120), Osl2 (LOC_Os04g52450),OsH36 (LOC_Os05g39770), OsWRKY23 (LOC_Os01g53260), OsWRKY72(LOC_Os11g29870), CDKA1 (LOC_Os03g02680), CAK1 (LOC_Os06g07480),CAK1A (LOC_Os06g22820), CMC5 (LOC_Os02g55410), CYCT1(LOC_Os02g24190), CYCA2.2 (LOC_Os12g31810), CYCA2.3 (LOC_Os01g13260),CYCB2.2 (LOC_Os06g51110), AOX1a (LOC_Os04g51150), AOX1b(LOC_Os04g51160), APX1 (LOC_Os03g17690), APX2 (LOC_Os07g49400), SODB(LOC_Os06g05110), SODA1 (LOC_Os05g25850), catA (LOC_Os02g02400), catB(LOC_Os06g51150),OsEXPA1 (LOC_Os04g15840),OsEXPA2 (LOC_Os01g60770),OsEXPA5 (LOC_Os02g51040), OsEXPA10 (LOC_Os04g49410), OsEXPA32(LOC_Os08g44790),OsEXPB3 (LOC_Os10g40720),OsEXPB5 (LOC_Os04g46650),OsEXPB9 (LOC_Os10g40090), and OsEXPB11 (LOC_Os02g44108).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Wild-type (Nipponbare, left) and del1 plants(right) at the tillering stage.

Supplemental Figure S2. Statistical analysis of plant height from 30 to110 d between wild-type and del1 plants.

Supplemental Figure S3. Internode assessment of culms in the wild-typeand del1 plants.

Supplemental Figure S4. Mutation position of wild type, del1, and sixother varieties.

Supplemental Figure S5. The phenotype of wild-type and RNAi trans-genic plants.

Supplemental Figure S6. Protein sequence alignment of DEL1 andhomologs.

Supplemental Figure S7. Cell wall structure in wild-type and del1 roots

Supplemental Figure S8. Expression analysis of cell expansion-relatedgenes in culms.

Supplemental Table S1. The basic agronomic traits in wild-type and del1plants.

Supplemental Table S2. Segregation analysis of F2 phenotype fromcrosses of del1 and five crop strains (NPB, TN1, ZF802, NJ06, and 93-11).

Supplemental Table S3. Clusters of orthologous groups of proteins func-tional catalog of differentially expressed genes in del1 plants

Supplemental Table S4. Cell wall function and senescence related genesidentified by RNA-seq analysis as being differentially in del1 plants.

Supplemental Table S5. Primers used for PCR in this study.

ACKNOWLEDGMENTS

We are grateful to Dr. Yihua Zhou (Institute of Genetics and DevelopmentalBiology, Chinese Academy of Sciences) for help with the monosaccharide andcellulose analyses, Dr. Yong Hu (Capital Normal University) for help with theflow cytometric analysis, and Dr. Paul Knox (Centre for Plant Science, Univer-sity of Leeds, UK) for kindly providing the protocol for the immunofluores-cence assay.

Received October 24, 2016; accepted April 13, 2017; published April 28, 2017.

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