A Rice PECTATE LYASE-LIKE Gene Is Required for Plant

Growth and Leaf Senescence1[OPEN]

Yujia Leng 2, Yaolong Yang 2, Deyong Ren 2, 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 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 for Crop Gene Resources and Genetic
Improvement, 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, 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 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 for
Crop Gene Resources and Genetic Improvement, 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.)
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 a
mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histological
analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the
decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and
TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene
was 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 but
predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic
activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In
addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1
plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf
senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE
LYASE-LIKE genes.

1 The development of higher plants is accompanied by

This research was supported by the National Natural Science
Foundation of China (Grant No. 91435105, 3161143006, 91535205),
the complex processes of cell division and cell expansion
National Key Basic Research Program (grant no. 2013CBA014), and and finally the emergence of many specialized organs,
the “Science and technology innovation project” of the Chinese Acad- tissues, and cells. As part of cell development in plants,
emy of Agriculture Sciences. cell walls perform essential functions, such as providing
2
These authors contributed equally to the article. tensile strength, shape, and protection, as well as helping
* Address correspondence to dalizeng@126.com or qianqian188@ the cell to maintain its internal pressure (Cosgrove, 2005).
hotmail.com. In growing plants, the cell wall is both resistant to the
The author responsible for the distribution of materials integral to high turgor pressure that drives growth and also
the findings presented in this article in accordance with the policy flexible enough to yield and expand selectively in re-
described in the Instructions for Authors (www.plantphysiol.org) is:
sponse to that pressure (Cosgrove, 2005; Xiao et al., 2014).
Dali Zeng (dalizeng@126.com).
Yu.L., Y.Y., and D.R. performed research; L.H., L.D., Y.W., L.C.,
This process involves a series of molecular mechanisms,
Z.T., Y.G., L.Z., J.H., G.Z., Z.G., L.G., Yo.L., X.L., and Z.K. analyzed including disruption of the intermolecular adhesion,
the data; Yu.L. wrote the article; Yu.L., D.Z., and Q.Q. designed the polymer lysis, religation and rearrangements, and/or
research; D.Z. revised the article. cleavage of polymer glycosyl linkages by enzymes
[OPEN]
Articles can be viewed without a subscription. (McQueen-Mason and Cosgrove, 1995; Van Sandt et al.,
www.plantphysiol.org/cgi/doi/10.1104/pp.16.01625 2007; Anderson et al., 2010; Park and Cosgrove, 2012).
Plant PhysiologyÒ, June 2017, Vol. 174, pp. 1151–1166, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved. 1151
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Leng et al.
Pectins are major components of the plant primary
cell wall and middle lamella and have several functions,
including involvement in maintaining plant growth and
development, promoting cell-to-cell adhesion, providing
structural support in soft tissues, defense responses, and
influencing wall 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 most
complex polysaccharide family in the living world, being
composed of as many as 17 different monosaccharides
and having more than 20 different linkages (Bonnin
et al., 2014). According to the current understanding of
pectins at the structure/function level, there are two
conceptual models of structural modification during
plant growth (Atmodjo et al., 2013). First, the large
number 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 wall
networks (Vincken et al., 2003; Xiao et al., 2014). HG is
secreted in a highly methylesterified form and selec-
tively demethylesterified by pectin methylesterases
(PME; Hongo et al., 2012). The demethylesterified HG
can either form a rigid gel by Ca2+-pectate cross-linked
complexes, or become more susceptible to cleavage by
two classes of pectin-degrading enzyme, namely pec-
tin/pectate lyase (PL/PEL; EC 4.2.2.10; EC 4.2.2.2) and
polygalacturonase (PG; EC 3.2.1.15) (Wolf et al., 2009;
Hongo et al., 2012). Hence, the methylesterification
status of HG can regulate cellular growth and cell
shape, and affect plant growth and development (Wolf
et al., 2009; Peaucelle et al., 2011).
PELs, a family of endo-acting depolymerizing en-
zymes, are responsible for the a-1,4-glycosidic linkages
in demethylesterified HG by b-elimination and pro-
duces 4,5-unsaturated oligogalacturonides (OGs) at
their nonreducing ends (Palusa et al., 2007). PELs have
been extensively studied in plant pathogenic bacteria
such as Erwinia chrysanthemi, which causes soft-rot dis-
eases (Barras et al., 1994). In plants, multiple functions
have been identified for PEL, and there is some sug-
gestion that it is involved in pollen, anthers, pistils, and
developing tracheary elements (Wing et al., 1990; Rogers
et al., 1992; Wu et al., 1996; Kulikauskas and McCormick,
1997; Domingo et al., 1998; Milioni et al., 2001), fruit
softening and ripening (Dominguez-Puigjaner et al.,
1997; Medina-Escobar et al., 1997; Nunan et al., 2001; Pua
et al., 2001), lateral root emergence (Laskowski et al.,
2006), cotton fiber elongation (Wang et al., 2010), leaf
senescence (Wu et al., 2013), and susceptibility to plant
pathogens (Vogel et al., 2002). It has also been shown to
be expressed in a wide range of tissues (Palusa et al.,
2007; Sun and van Nocker, 2010). In addition, recent
research has revealed that the overexpression of aspen
PtxtPL1-27 can increase the solubility of wood matrix
polysaccharides (Biswal et al., 2014). The modification of
pectins may be important in the field of biotechnology
for improving woody biomass (Biswal et al., 2014).
1152
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
The large family of PECTATE LYASE-LIKE (PLL)
genes all exhibit redundant or unique functions in plant
evolution, and this diversity may enhance plasticity in
adaptation to changing environments (Sun and van
Nocker, 2010). In rice (Oryza sativa), genome prediction
has suggested that there are 14 PLL genes, but only one
of these (ospse1) has been analyzed genetically (Wu
et al., 2013). In this study, we report on the isolation and
characterization of a recessive mutant, dwarf and early-
senescence leaf1 (del1), in rice. The mutation of DEL1 led
to a phenotype involving abnormal plant growth and
leaf senescence. DEL1 encodes a PEL precursor and is a
member of a multigene family in rice. The loss of func-
tion of DEL1 decreased total PEL activity, increased the
degree of methylesterified HG, and perturbed cell wall
composition 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 in
expression in the del1 plants. Our findings suggest that
DEL1 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 ethyl
methanesulfonate-mutagenized japonica cultivar, Nip-
ponbare. del1 exhibited dwarfism at the seedling stage (5 d
after germination), and its main root length was clearly
reduced, which reached 47.8% of that in the wild type
(Fig. 1, A and C). Meanwhile, the number of lateral roots
in del1 decreased significantly to just 44.7% of that in the
wild type (Fig. 1, B and D). The plant height of del1 was
also shorter than that of the wild type throughout the
growth period, being only about 36.8% of that of the wild
type at the mature stage (Fig. 1, E and F; Supplemental
Figs. S1 and S2), and the internode length, thickness, and
diameter of del1 were significantly reduced (Supplemental
Fig. S3). Compared with the wild type, the tiller number
and panicle length were also clearly reduced in del1 plants
(Fig. 1, E, G, H, and K). Moreover, grain development also
changed significantly, with grain size and grain weight
being diminished in del1 plants (Fig. 1, I, J, and L;
Supplemental Table S1). Besides these phenotypic
findings, the heading date of del1 was also notably
retarded, along with decreases in leaf length and leaf
width (Supplemental Table S1). These phenotypes
clearly suggest that plant size was significantly lower
in del1 plants.
Cell Number Is Reduced in del1 Plants
The size of a plant organ is determined by its cell size
and number of cells, which are related to cell expansion
and cell division, respectively (Krizek, 2009). To deter-
mine the causes of organ size reduction in del1 plants,
we conducted microscopic observation on the second
Plant Physiol. Vol. 174, 2017

culms and compared the findings between the wild
type and del1 using paraffinized sections. Cross sections
of culms revealed that the size of sclerenchyma cells in
del1 was less than that in the wild type (Fig. 2, A–D).
Statistical analysis showed that the cell number of del1 was
only 88.56% of that in the wild type (Fig. 2E). Moreover,
the number of layers of sclerenchyma cells in del1 was
reduced by two (Fig. 2, C, D, and F). Longitudinal sections
of culms in del1 displayed a significant change in cell size
(Fig. 2, G and H). The cell length in del1 was 59.6% longer,
and the cell width was 20.8% narrower than that in the
wild type (Fig. 2, I and J). Further investigation of the total
number of parenchyma cells showed that del1 had only
19% 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 in
del1 was affected by the cell cycle, we further investi-
gated the cell cycle progression by flow cytometry. The
results of the suspension cell lines of del1 revealed a
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DEL1 Affects Rice Growth and Leaf Senescence
Figure 1. Comparison of phenotype
between wild-type and del1 plants. A,
Wild-type (Nipponbare, left) and del1
plants (right) 5 d after sowing. Scale
bar = 2 cm. B, Root length of wild-type
(left) and del1 plants (right) 5 d after
sowing. Scale bar = 1 cm. C and D,
Statistical analysis of root length and
lateral root number between wild-type
and del1 plants. Twenty plants were mea-
sured. Error bars indicate SD; **P , 0.01
(Student’s t test). E, Wild-type (left) and del1
plants (right) at maturity. Scale bar = 10 cm.
F and G, Statistical analysis of plant height
and tiller number between wild-type and
del1 plants. Twenty plants were measured.
Error bars indicate SD; **P , 0.01 (Stu-
dent’s t test). H, Phenotype of panicle
between wild-type (left) and del1 (right)
plants. Scale bar = 5 cm. I, Floret with
the lemma removed, wild-type (left)
and del1 (right). Scale bar = 0.5 cm. J,
Mature seed and brown rice of wild
type (left) and del1 (right). Scale bar =
0.5 cm. K and L, Statistical analysis of
panicle length and thousand grain
weight between wild-type and del1
plants. Twenty panicles were mea-
sured. Error bars indicate SD; **P , 0.01
(Student’s t test).
significant increase in the number of cells in the G1
phase and decreases in the S and G2/M phases of the
cell cycle, implying that the cell cycle was delayed at the
G1 phase (Fig. 3, A–C). We also analyzed the expression
of cell-cycle-related genes in wild-type and del1 plants
using real-time reverse transcription (RT)-PCR. The
expression levels of genes related to the G1 phase of the
cell cycle, CDKA1, CAK1, CAK1A, MCM5, and CYCT1,
were down-regulated by ;10% to 45%, while little dif-
ference was observed for genes related to the G2 phase of
the cell cycle in del1 mutants (Fig. 3D). Therefore, we
conclude that the mutation of DEL1 delays cell cycle
progression 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 leaf
margin exhibited a faint yellow color from 5 d after
germination (Fig. 4A). With the development of plants,
1153
Leng et al.

Figure 2. Histological characteriza-

tion of culms in wild-type and del1
plants. 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, Statistical
analysis of cell number and scleren-
chyma cell layer number between
wild-type and del1 plants, means 6
SD of five independent replicates. G
and 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 and
cell width between wild-type and
del1 plants, mean 6 SD of 30 cells.
**P , 0.01 (Student’s t test). K,
Number of parenchyma cells (PCs) for
internode II of wild-type and del1
plants, mean 6 SD of five independent
replicates.

the young leaves were pale white and then turned
green upon maturation, while the old leaves exhibited
withering and cracking (Fig. 4, B–D). The chlorophyll
content in del1 plants was significantly lower, being only
44.0% of that in the wild type (Fig. 4E). In addition, trans-
mission electron microscopy (TEM) revealed the presence
of well-developed mesophyll cells and membrane-intact
chloroplasts in fully developed wild-type leaves, whereas
the disordered arrangement of grana thylakoid and the
degradation of chloroplasts was observed in del1 plants
(Fig. 4, F and G). Moreover, the photosynthetic rate in
del1 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 of
expression of many genes, including those for transcrip-
tion factors (Wang et al., 2015). To confirm senescence in
the del1 plants, the expression levels of senescence-
associated genes (SAGs) and related transcription
factors (Osl2, OsH36, SGR, OsNAC2, OsWRKY23,
and OsWRKY72) were determined by real-time RT-PCR.
The expression levels of Osl2, OsH36, SGR, OsNAC2,
1154
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
OsWRKY23, and OsWRKY72 mRNAs were 2.0 to 8.0
times higher than in wild-type leaves (Fig. 4I). The
up-regulated expression patterns of SAGs and tran-
scription factors further support the notion that early
leaf senescence occurred in del1 plants.
ROS Accumulation and Programmed Cell Death Are
Enhanced in del1 Plants
The accumulation of ROS can lead to leaf senescence
(Khanna-Chopra, 2012). Therefore, we performed nitro
blue tetrazolium (NBT) staining and 3,39-diaminobenzidine
(DAB) staining tests to detect O22 and H2O2 accumulation,
respectively. Extensive NBT staining was observed in
del1 plants, whereas the staining was minimal in wild-
type leaves (Fig. 5A). A brown color was seen for the
DAB 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 accumulated
in del1 plants. During senescence, plant cell membrane
damage can lead to a change in electrolyte leakage
(Blum and Ebercon, 1981). In del1 plants, the electrolyte
Plant Physiol. Vol. 174, 2017

Figure 3. Cell cycle analysis of wild-type and del1 plants. A and B, Flow
karyotype histogram of wild-type (A) and del1 (B) leaves. C, Quantification
of the DNA profiles of wild-type and del1 plants. D, Relative expression
levels of cell cycle-related genes in wild-type and del1 plants, mean 6 SD of
three independent replicates. *P , 0.05, **P , 0.01 (Student’s t test).
leakage was 45.8% higher than that in the wild type (Fig.
5C), suggesting that del1 lost more membrane integrity
during 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 of
these enzymes. The results indicated that the activities of
superoxide dismutase (SOD) and peroxidase (POD) in-
creased in del1 plants, at 100.3 and 110.4 U/mg fresh
weight in del1 leaves, which were much higher than the
levels of 62.2 and 102.3 U/mg fresh weight in the wild-type
leaves, respectively (Fig. 5, D and E). In the process of leaf
senescence, ROS-scavenging systems may play an
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DEL1 Affects Rice Growth and Leaf Senescence
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 of
ROS-scavenging-related genes (AOX1a, AOX1b, APX1,
APX2, SODB, SODA1, catA, and catB) were 2.5 to 13.6
times 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 staining
revealed that the leaf apex and leaf margin of del1 were
colored blue, but the counterpart wild type remained green
(Fig. 5G), suggesting that some of the cells in the leaf apex
and leaf margin of the del1 plant were dead. One basic
feature of PCD is the condensation of nuclear chromatin,
which is caused by endonucleolytic degradation of nuclear
DNA (Simeonova et al., 2000). We accordingly used the
TUNEL assay to determine whether the del1 plants in-
duced PCD. A few of the nuclei in the wild type were
TUNEL positive; in contrast, most nuclei in the del1 leaf
sections were TUNEL positive (Fig. 5, H–K), indicating that
DNA 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 employed
to isolate the corresponding gene. The mapping popu-
lation was generated by crossing DEL1 with Taichung
Native 1, a wild-type indica variety with DNA poly-
morphism with japonica. All the F1 plants exhibited a
normal phenotype matching that of the wild type. In
the F2 segregating populations, normal and mutant
phenotypes showed a typical segregation ratio of 3:1
(Supplemental Table S2). This finding suggests that del1
is controlled by a single recessive nuclear gene.
Thirty F2 plants with the del1 phenotype were used for
primary mapping, and DEL1 was found to be located on
the long arm of chromosome 10 between markers M1
and M14. Furthermore, by using 1081 homozygous
mutant plants, DEL1 was further narrowed down to
an ;45-kb region with molecular markers as shown in
Supplemental Table S5. This 45-kb region is in the BAC
AC025905 and contains eight putative open reading
frames (ORFs), as annotated by the MSU Rice Genome
Annotation Project (Fig. 6A). Upon sequencing analysis
of these eight ORFs, one base pair mutation was found at
position 1940 of ORF LOC_Os10g31910. This change
from G to T causes a substitution at the 365th amino acid
residue from Trp to Leu (Fig. 6B). By examining this base in
six other rice varieties, we found that they all exhibited the
wild-type genotype (Supplemental Fig. S4).
To confirm that LOC_Os10g31910 is responsible for the
del1 phenotype, a 7843-bp wild-type DEL1 DNA fragment
containing the promoter region and the entire ORF was
introduced into del1 plants by Agrobacterium tumefaciens-
mediated transformation. Analysis of independent
transgenic plants by PCR using primers flanking the
mutation site, together with further sequencing, revealed a
1155
Leng et al.

Figure 4. Leaf phenotype and identifica-

tion of leaf senescence in DEL1. A to D,
Leaf phenotype from young (A), tillering (B
and C), and heading (D) stages. Scale
bars = 1, 5, 5, and 1 cm in A, B, C, and
D, respectively. E, Statistical analysis of
chlorophyll content between wild-type
and del1 plants, mean 6 SD of five inde-
pendent replicates. **P , 0.01 (Student’s
t test). F and G, Transmission electron
microscopy analysis of senescence leaves
of wild-type (F) and del1 plants (G). Scale
bar = 0.5 mm. H, Statistical analysis of
photosynthesis rate between wild-type
and del1 plants, mean 6 SD of five inde-
pendent replicates. **P , 0.01 (Student’s
t test). I, Relative expression levels of se-
nescence-related genes and transcription
factors in wild-type and del1 plants,
mean 6 SD of three independent repli-
cates. *P , 0.05, **P , 0.01 (Student’s
t test).

double peak (G and T) at the mutation site, confirming the
presence of normal DEL1 gene sequence (Fig. 6C). The
transgenic plants rescued the phenotypes of del1, whereas
in all of those transformed by pCAMBIA1300, there was a
failure to rescue the mutant phenotype, confirming that
disruption of the DEL1 gene was responsible for the del1
mutant 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). The
expression of DEL1 was significantly decreased in RNAi
plants (Supplemental Fig. S5F). Therefore, we conclude
that LOC_Os10g31910 is the DEL1 gene.
DEL1 Encodes a Pectate Lyase Precursor
Sequence analysis indicated that DEL1 cDNA is 1476 bp
in length and encodes a protein of 491 amino acid residues
(Fig. 7A). A search using SignalP and Pfam revealed that
the DEL1 protein contains a 28-amino acid signal peptide
and one predicted PelC domain (Fig. 7A). Multiple
1156
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
sequence alignment analyses revealed that DEL1 contains
all the conserved residues of PELs known to be involved
in Ca2+ binding, substrate binding, and catalysis and has
stronger similarity with plant PelC than Erwinia chrys-
anthemi (Supplemental Fig. S6).
An unrooted phylogenetic tree was built among PEL
members of rice, Arabidopsis (Arabidopsis thaliana), and
seven known plant PELs using the neighbor-joining
method, which revealed that PELs were divided into
five major clades, with clade I being further divided
into four subclades (Fig. 7B). DEL1 belongs to Ic and is
in 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 expression
of the DEL1 genes, we isolated RNA from different
tissues, including young roots, young sheaths, young
leaves, mature roots, mature sheaths, mature leaves, culms,
Plant Physiol. Vol. 174, 2017

Figure 5. ROS accumulation and enhancement of PCD in wild-type and
del1 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 electrolyte
leakage (C), SOD activity (D), and POD activity (E) in leaves between wild-
type and del1 plants, mean 6 SD of five independent replicates. **P , 0.01
(Student’s t test). F, Relative expression levels of ROS detoxification-related
genes 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 in
wild-type (left) and del1 plants (right). H to K, TUNEL assay of leaves. DAPI
staining of wild-type (H) and del1 plants (J). Positive results of wild-type (I)
and del1 plants (K). Scale bar = 50 mm.
panicles, and spikelets, and determined the transcript
levels of the DEL1 genes by real-time RT-PCR. The results
revealed that DEL1 was ubiquitously expressed in all the
tissues examined at the young and mature stages, and high
levels of DEL1 expression were observed in elongating
tissues, 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
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DEL1 Affects Rice Growth and Leaf Senescence
of the native promoter of the DEL1 gene. Six indepen-
dent DEL1:GUS transgenic lines were analyzed, and all
of them exhibited similar results. The GUS activity was
expressed in all of the tissues and was consistent with
the qRT-PCR data (Fig. 8, B–J).
Mutation of DEL1 Decreased Total PEL Activity and
Altered Cell Wall Composition and Structure
Since PelC is the only domain identified in DEL1, it
may be critical for its function. Therefore, we deter-
mined the total PEL activity. As expected, PEL enzyme
activity was significantly decreased in del1 plants. Es-
pecially in culms, the PEL activity was only 32.0% of
that in the wild type (Fig. 9A). This result is consistent
with the real-time RT-PCR (Fig. 8A) and the ProDEL1:
GUS expression results (Fig. 8, B–J).
The morphological phenotypes suggest that the cell
wall structure in the mutant plants may also be altered.
We therefore analyzed the structure of wild-type and
del1 plants by TEM. The results revealed that the wall
thicknesses of bundle sheath fiber cells in del1 culms
were altered (Fig. 9, D–G). Specifically, the thicknesses of
the middle lamella and secondary cell wall were reduced
by 51.6% and 40.6%, respectively, and that of the pri-
mary cell wall was 15.6% higher than those of the wild
type (Fig. 9, H–J). Similarly in roots, the thicknesses
(sclerenchyma cell) of the middle lamella and secondary
cell wall were reduced by 18.5% and 41.4%, respectively,
and that of the primary cell wall was 12.7% higher than
those of the wild type (Supplemental Fig. S7).
To determine whether changes in the cell wall
structure affected the wall composition, we analyzed
the cellulose, hemicellulose, and pectin contents and
compared them between wild-type and del1 culms. The
cellulose content of del1 was reduced by ;20%, while
the contents of hemicellulose 1 (HC 1), hemicellulose
2 (HC 2), and pectin were increased by 58.3, 27.3, and
117.2%, respectively (Fig. 9B). In addition, the levels of
seven neutral monosaccharides were also determined
and compared between the wild-type and del1 plants.
All the neutral monosaccharides in del1 exhibited sig-
nificant increases, except for Xyl (Fig. 9C). These results
suggest that the DEL1 mutation causes complex com-
positional and structural alterations in cell walls.
Mutation of DEL1 Increased the Degree of
HG Methylesterification
Given that PEL can catalyze the a-1,4 glycosidic
linkages of demethylated HG by b-elimination, we used
immunohistochemical techniques to discern in situ as-
pects of cell wall microstructures and locate polymers
precisely. The second culms from wild-type and del1
plants were probed using JIM5, LM18, and LM19 anti-
bodies, which are used to recognize partially demethy-
lesterified and unesterified HG; JIM7 antibody, which
labels moderately high extent of methylesterified HG;
and 2F4 antibody, which binds to HG with degrees of
methylesterification up to 40% (Verhertbruggen et al., 2009;
1157
Leng et al.

Figure 6. Map-based cloning and iden-

tification of DEL1. A, Fine mapping of
DEL1. The del1 locus was mapped to
a 45-kb region on chromosome 10. B,
Schematic diagram of DEL1. Black rect-
angles represent exons. Black inverted
triangle represents mutant site. C, Se-
quencing analysis of the DEL1 transcripts
in T0 transgenic lines. D and E, Phenotype
of the complementation transgenic line:
Wild type (left), complementation trans-
genic line (middle), and empty vector
control (right). Scale bars = 10 cm and
4 cm, respectively. F, Expression levels of
DEL1 detected by qRT-PCR in wild-type
and transgenic plants, mean 6 SD of three
independent replicates. G to I, Statistical
analysis of plant height (G), tiller number
(H), and flag leaf length (I) in wild-type
and transgenic plants, mean 6 SD of
10 independent replicates.

Held et al., 2011). As shown in Figure 10, immunolabeling of analysis. A total of 491 differentially expressed genes
culm sections with JIM7, LM18, LM19, and 2F4 indicated an (DEGs) were found with a P value , 0.05 (Supplemental
apparent increase in the recognition of HG epitopes, while Table S3). Among these genes, 332 were up-regulated and
JIM5 exhibited a slight increase, suggesting that the degree 159 were down-regulated in del1 plants (Supplemental
of methylesterified HG was higher in del1 plants. Table S3). Clusters of orthologous groups of proteins
functional catalog showed that in DEGs were assigned to
Activation of Cell Wall- and Senescence-Related Genes 39.2% and 50.9% of the up- and down-regulated genes,
Revealed by Transcriptome Analysis respectively (Supplemental Table S3). Most of the genes
up-regulated in del1 were involved in secondary metab-
To further unravel the function of DEL1 in rice, 10-d-old olite biosynthesis, transport, and catabolism, carbohy-
wild-type and del1 plants were used for transcriptome drate transport and metabolism, and lipid transport and
1158 Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
 DEL1 Affects Rice Growth and Leaf Senescence

metabolism (Supplemental Table S3). The majority of the

down-regulated genes were associated with translation,
ribosomal structure and biogenesis, secondary metabo-
lites biosynthesis, transport, and catabolism, and cell
wall/membrane/envelope biogenesis (Supplemental
Table S3). Of note, a set of the DEGs associated with
cell wall and senescence were significantly altered in
del1 plants, such as the PME gene (OsPME1), b-expansin
gene (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). These
results suggested that the mutation of DEL1 might affect
the expression of cell wall- and senescence-related genes
in an indirect manner.

Figure 8. Expression analysis of DEL1. A, Transcription level of DEL1 in

various organs, mean 6 SD of three independent replicates. YR, Young
root; YL, young leaf; YS, young sheath; MR, mature root; C, culm; ML,
mature leaf; MS, mature sheath; P, panicle; S, spikelet. B to J, GUS
analysis 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 =
500 mm; E, lateral root, scale bar = 250 mm; 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, scale
bar = 500 mm.

Figure 7. Prediction of the primary sequence and phylogenetic
analysis 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 domain
is 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 other
plants. The numbers at each node represent the bootstrap support
(percentage), and scale bar is an indicator of genetic distance based
on branch length.
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DISCUSSION
PEL is an endogenous pectin-degrading enzyme that
is capable of cleaving a-1,4-glycosidic linkages in
demethylated pectin by b-elimination (Palusa et al.,
2007). It is a ubiquitous enzyme in higher plants and is
encoded by at least 26, 22, and 14 genes in Arabidopsis,
poplar (Populus trichocarpa), and rice, respectively (Palusa
et al., 2007). Although several PLL genes are considered to
play potentially diverse physiological roles in plants, such
as 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 model
monocot rice using a forward genetic approach; it con-
tains a PelC domain and represents a multigene family in
1159
Leng et al.

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

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 methylesterified
HG, 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 dual
molecular function of PLL genes in rice.
DEL1 Impacts Plant Growth by Regulating Cell
Cycle Progression
Cell division/expansion is a fundamental, dynamic
cellular process driving plant growth and develop-
ment, and it enables the plant and various organs to
develop to suitable sizes (Duan et al., 2012). The orderly
development 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
1160
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
studies have uncovered some key regulators and genes
that affect plant organ size, the intrinsic mechanisms
responsible for organ size variation are yet to be fully
understood (Krizek, 2009; Duan et al., 2012). In this
study, we identified a PLL gene, DEL1, that appeared to
play an important role in the control of organ size in
rice. In del1 plants, various organs were dramatically
reduced in size, including in terms of root length, leaf
size, plant height, grain size, and panicle and internode
length (Fig. 1; Supplemental Table 1). Inside the organs,
paraffinized sections of internodes revealed that cell
number was lower in the del1 plants (Fig. 2), suggesting
that cell division is inhibited in the mutant.
Cell division requires a range of complicated pro-
cesses that must be strictly executed in a spatially and
temporally controlled manner (Dewitte and Murray,
2003). The inhibition of cell division could alter cell cycle
progression and affect plant development (Dewitte and
Plant Physiol. Vol. 174, 2017
 DEL1 Affects Rice Growth and Leaf Senescence

Figure 10. Immunohistochemical localization of HG in culm sections of wild-type and del1 plants. A to T, Immunolocalization of
HG of wild-type (A and C) and del1 plants (B and D) with JIM7, wild-type (E and G) and del1 plants (F and H) 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.

Murray, 2003). Our study demonstrates that the cell
cycle in the G1 phase was significantly retarded in del1
plants (Fig. 3, A–C). Recent evidence has indicated that a
variety of cell cycle regulators play roles as targets cou-
pling cell proliferation with development, and their
proper functioning is crucial for patterning (Ramirez-
Parra et al., 2005). Consistent with the flow cytometric
results, the expression levels of genes regulating the G1
phase of the cell cycle were down-regulated in del1, and
no 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 through
ROS Accumulation
Leaf senescence is a complex process that involves
many highly organized molecular and cellular changes,
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
such as the disintegration of chloroplasts; down-
regulation of photosynthesis; degradation of nucleic
acids, proteins, and lipids; and recycling of nutrients (Lim
et al., 2007). This process is controlled by both internal and
external factors. To date, although many SAGs and/or
transcription factors have been identified, the complex
mechanisms responsible for leaf senescence in rice remain
poorly understood. Recently, Wu et al. (2013) identified
the novel leaf senescence gene OsPSE1, which encodes a
PEL. 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 leaf
senescence. TEM observation, physiological analysis, and
TUNEL assays showed that leaf senescence was induced
in del1 plants, resulting in the withering and cracking of
leaves (Fig. 4). Moreover, the up-regulated expression
levels of SAGs and transcription factors also demonstrate
leaf senescence in del1 plants (Fig. 4I).
1161
Leng et al.
ROS are byproducts of various metabolic processes
such 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 assume
several important roles in leaf senescence (Wang et al.,
2015). In the present study, staining by NBT and DAB
indicated the accumulation of O22 and H2O2 in del1
plants (Fig. 5, A and B), and there was also an elevated
level of electrolyte leakage, which is a marker of cell
membrane damage (Fig. 5C). Meanwhile, the activities
of SOD and POD were increased in del1 plants (Fig. 5,
D and E). These results indicate that leaf senescence in
del1 plants is triggered by the accumulation of ROS.
Under normal physiological conditions, cells control
ROS levels by balancing the generation of ROS with
their elimination by the ROS scavenging system, but
the overproduction of ROS can trigger retrograde
signaling from chloroplasts to the nucleus, resulting in
alteration of the expression of nuclear-encoded genes
(Tan et al., 2014). Consistent with this, the expression
of ROS-scavenging genes was significantly up-regulated
(Fig. 5F). In addition, transcriptome analysis also revealed
that a set of the DEGs associated with senescence was
significantly altered in del1 plants (Supplemental
Table S4).
Taking these findings together, it is clear that ROS
accumulation-mediated chloroplast membrane break-
age and chloroplast degradation play an important role
in leaf senescence in the del1 plants.
Possible Molecular Mechanism of DEL1 Responsible for
the Mutant Phenotypes
PLL genes have been shown to exhibit a broad range
of functions in plants, which depend on the degrada-
tion of demethylesterified HG and alteration of cell
wall composition and structure (Yadav et al., 2009;
Biswal et al., 2014). In the process of fruit ripening, the
cell wall needs to loosen the xyloglucan-cellulose net-
work and pectin solubilization, this processes increasing
the access of degradative enzymes to their substrates
(Brummell, 2006). For pathogen defense, the accumu-
lation of pectin increases hydrogen bonding in the
extracellular matrix, resulting in decreased nutrient
availability to the pathogen (Vogel et al., 2002). The
decrease of de-esterified pectin in cotton fiber can
promote its elongation (Wang et al., 2010). Our study
also demonstrates that the mutant phenotype of del1
depends on the alteration of cell wall composition and
structure. Despite our failure to purify the fusion DEL1
protein via prokaryotic expression in Escherichia coli
or transient expression in Nicotiana benthamiana (data
not shown), the mutant phenotype indicated that
DEL1 protein plays an important role in rice growth
and leaf senescence.
Many cell wall-related mutants display abnormal
growth and morphogenesis (Pien et al., 2001). It has been
speculated that cell wall biogenesis and modification are
1162
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
tightly associated with cell growth via key genes at the
interface of morphogenesis, the cell cycle, and cell wall
biogenesis (Somerville et al., 2004; Zhang et al., 2010).
In rice, the Brittle Culm 12 gene, encoding a kinesin-4
protein, has been implicated in cell cycle progression,
cellulose microfibril deposition, and wall composition
(Zhang et al., 2010). In our study, the del1 mutant
exhibited cell wall abnormalities and the retardation of
cell cycle progression, which indicates that DEL1 is at the
interface between the cell cycle and cell wall biogenesis.
The dynamic interactions of plant cells depending on
the status of pectin in the cell wall form an important
regulatory mechanism of growth and development
(Wolf et al., 2012). The degree of HG methylester-
ification has a large effect on the physical properties of
the cell wall (Held et al., 2011). A high degree of HG
methylesterification impedes the formation of calcium-
mediated cross linking complexes, which is thought to
promote cellular expansion by increasing wall flexibil-
ity (Wolf et al., 2009). Our findings revealed that the
degree of methylesterified HG was increased, as de-
termined by immunohistochemical assay, which indi-
cates that the cell wall loosened and expanded in the
del1 plants (Fig. 10). Consistent with this, the cell length
was significantly increased and the expression levels of
expansion were all dramatically up-regulated in the
del1 plants (Fig. 2, G and H; Supplemental Fig. S8).
These results indicate that the function of DEL1 affects
not only cell number, but also cell expansion.
In addition, our findings also revealed an early leaf
senescence phenotype in comparison to that in wild-
type plants. This initially appears paradoxical, but it
could potentially be explained by the alterations of cell
wall structure. Pectin OGs are oligomers of a-1,4-linked
galacturonosyl residues and are released by PEL- and
PG-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 be
cleaved by DEL1 and other PELs or PGs. The alternative of the cell wall
regulated the cell cycle/expansion and ROS, enabling normal rice
growth and leaf senescence process.
Plant Physiol. Vol. 174, 2017

Oligogalacturonides can produce ROS signal molecules
and trigger many abiotic stress responses in plants
(Ferrari et al., 2013). In our study, mutation in DEL1
significantly increased ROS content. We speculate that
DEL1 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 and
structure might act as downstream components of the
regulatory networks that control cell cycle and expan-
sion, as well as ROS, enabling normal growth and leaf
senescence in rice (Fig. 11).
MATERIALS AND METHODS
Plant Materials
The rice (Oryza sativa) del1 mutant 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 an
indica cultivar, Taichung Native 1 (TN1). All plants were cultivated in paddies in
Hangzhou (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 glacial
acetic acid, and 3.7% formaldehyde overnight at 4°C, then dehydrated with a
graded series of ethanol, infiltrated with xylene, and embedded in paraffin
(Sigma-Aldrich). The specimens were sectioned (8 mm thickness) with a Leica
RM2245, transferred onto glass slides with poly-L-lysine-coated, deparaffinized
with 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 least
4 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 ethanol
series, and infiltrated with Suprr Kit (Sigma-Aldrich). The specimens were then
sectioned (70 nm ultrathin) with a Leica EM UC7 ultratome, and the sections
were stained by uranyl acetate and alkaline lead citrate for 10 min before being
observed by TEM with a Hitachi model H-7650.
Flow Cytometric Analysis
Cell cycle analysis was performed in accordance with the method described
by Galbraith et al. (1983). In brief, suspension cells were cut using a razor blade
and 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 then
passed through a 20-mm mesh to remove cellular debris. After staining with
49,6-diamino-phenylindole (DAPI; 2 mg mL21), the nuclei were analyzed using
FACSAria II flow cytometer (BD Biosciences). A total of 30,000 events were
recorded, and three independent flow cytometric experiments were carried out
and analyzed using the ModFit LT (Verity Software House) and FlowJo V10
software (Tree Star).
Chlorophyll Content and Net Photosynthesis Rate
A total 0.2 g of samples from wild-type and del1 leaves were extracted with
80% acetone. The chlorophyll content was determined according to the method
previously described (Yang et al., 2016). Net photosynthesis rate was measured
using a Walz GFS-3000 (Eichenring). Parameter settings were accordance with
the manufacturer’s instructions.
Histochemical Staining and ROS-Scavenging
Enzyme Assays
DAB and NBT staining was used to detect the accumulation of ROS, as
previously described (Blum and Ebercon, 1981). Trypan blue staining was
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DEL1 Affects Rice Growth and Leaf Senescence
performed as described previously (Koch and Slusarenko, 1990). Electrolyte
leakage was determined in accordance with a previous study (Zhou and Guo,
2009). Assays of the activities of SOD and POD were conducted in accordance
with 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 containing
0.1% 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 Situ
Cell Death Detection Kit (Roche). The green fluorescence of fluorescein and blue
fluorescence of DAPI was analyzed using a Carl Zeiss LSM 710 laser-scanning
confocal microscope (Gottingen).
Gene Cloning and Complementation
For genetic analysis, 1081 F2 mutant plants were used for fine mapping
of the DEL1 locus. The molecular markers of polymorphism are listed in
Supplemental Table S5. The markers were designed using Primer 5 software
(Primer-E). Gene prediction and sequence analysis were performed using the
publicly 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 genomic
DNA fragment containing the entire DEL1 coding region, a 1,998-bp upstream
sequence, and an 841-bp downstream region was amplified using KOD FX
(Toyobo) from Nipponbare genomic DNA and inserted into the binary vector
pCAMBIA 1300. The recombinant binary vector was introduced into Agro-
bacterium tumefaciens strain EHA105 by electroporation and transformed into
the del1 mutant callus as described previously (Hiei et al., 1994). For the RNAi
construct, a 336-bp fragment was amplified by PCR from Nipponbare cDNA
and inserted into the vector pTCK303. The recombinant binary vector was
transformed into the Nipponbare callus. The primer sequences used in this
study 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 SignalP
version 4.1 (Petersen et al., 2011). Homologous protein sequences of DEL1
were 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). A
phylogenetic tree was constructed using MEGA4 software (http://www.
megasoftware.net/).
Gene Expression Analysis
Total RNA from roots, culms, sheaths, leaves and panicles at young and
mature stages was extracted using the RNeasy plant mini kit (Qiagen). RNA
reverse transcription was performed using ReverTra Ace real-time PCR-RT kit
with gDNA remover (Toyobo). After synthesis, the cDNA reaction was diluted
five times in TE buffer, and 1 mL was used for real-time RT-PCR using the SYBR
Green PCR Master Mix kit (Applied Biosystems) and gene-specific primers
(Supplemental Table S5) in ABI7900 (Applied Biosystems). The tissue-level
expression pattern of DEL1 was analyzed using the GUS reporter gene. The
promoter of DEL1 (2,038 bp upstream of ATG) was amplified from the genome
DNA of Nipponbare and inserted into the binary vector pCAMBIA1305
in-frame with the GUS reporter gene. The binary vector was introduced into the
Nipponbare callus to generate transgenic plants. The positive transgenic plants
were used to analyze the GUS reporter gene. Different tissues of T1 plants were
incubated 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 examine
GUS 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 modified
version of the method of Collmer et al. (1988). A total of 0.5 g of sample was
suspended in 1.5 mL of extraction buffer (20 mM sodium phosphate, pH 7.0,
1163
Leng et al.
20 mM Cys/HCl, 1% polyvinylpyrrolidone, MW 360,000, and 1 mM phenyl-
methylsulfonyl fluoride) and centrifuged at 10,000g for 30 min. The supernatant
was 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 reaction was incubated at 37°C for 30 min, and then stopped by the
addition of 9% ZnSO4$7H2O and 0.5 M NaOH. The control tubes received the
enzyme after the addition of ZnSO4$7H2O and NaOH. One unit of PEL activity
was defined as the amount of enzyme that produces 1 mM of 4,5-unsaturated
product in 1 min under the assay conditions. The activity of PEL is expressed in
units per mg of protein. Soluble protein concentrations were determined in
accordance 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 were
fixed and embedded in glycol methacrylate. Two-micrometer-thick sections
were cut with a Leica RM2265. Immunolabeling was performed in accordance
with 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 as
controls to assess the autofluorescence of the samples. The labeled sections were
observed using Carl Zeiss LSM 710 confocal microscope.
Compositional Analysis of Neutral Glycosyl Residues and
Cellulose Content
Alcohol-insoluble residues of culms were prepared as described by Zhang
et 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 were
centrifuged to collect the supernatants, air-dried, and then dissolved with a 1 M
ammonium hydroxide buffer containing NaBH4. The alditol acetate derivatives
were analyzed using an Agilent 7890 GC system equipped with a 5975C MSD
(gas chromatography-mass spectrometry; Song et al., 2013). To measure the
crystalline cellulose content, the pellets remaining after trifluoroacetic acid
treatment were hydrolyzed with Updegraff reagent (Updegraff, 1969). The
samples were subsequently centrifuged and the supernatant removed. The
pellets were then treated with 72% sulfuric acid. The cellulose content was
quantified via an anthrone assay, and three replicates were included.
Cell Wall Extraction and Measurement of
Polysaccharide Content
Cell wall materials and fractionation components were analyzed in accor-
dance with the methods of Zhong and Lauchli (1993). The samples were ground
in liquid nitrogen to a fine power and suspended in 75% ethanol for 25 min in an
ice-cold water bath. The samples were then centrifuged at 12,000 rpm for 15 min
and removed the supernatant. Next, the pellet was suspended and washed with
precooled acetone, followed by methanol:chloroform and then with methanol.
The remaining pellet as a crude cell wall fraction was freeze-dried and stored at
4°C for future use.
The cell wall extracts were fractionated into three parts: pectin, HC 1, and HC
2. First, the pectin fraction was extracted twice using 0.5% ammonium oxalate
buffer containing 0.1% NaBH4 (pH 4) in a boiling water bath for 1 h. Next, the
pellets were subjected to triple extractions with 4% KOH containing 0.1%
NaBH4 at room temperature for 8 h each, followed by a similar extraction with
24% KOH containing 0.1% NaBH4. The HC 1 and HC 2 fractions were referred
to as hemicellulose material (Yang et al., 2008). The uronic acid content in the
pectin fraction was assayed in accordance with the method of Blumenkrantz
and 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 for
transcriptome sequencing, and each sample was pooled for total RNA isolation
with three biological replicates. Total RNA was extracted using TRIzol reagent
(Invitrogen Life Technologies). Transcriptome sequencing was performed in
Biomarker Biotechnology Corporation using the Illumina system HiSeq2500
1164
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
(Illumina) according to the standard procedure. Transcriptome assembly was
performed 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 of
DEGs 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 data
libraries 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 to
110 d between wild-type and del1 plants.
Supplemental Figure S3. Internode assessment of culms in the wild-type
and del1 plants.
Supplemental Figure S4. Mutation position of wild type, del1, and six
other varieties.
Supplemental Figure S5. The phenotype of wild-type and RNAi trans-
genic plants.
Supplemental Figure S6. Protein sequence alignment of DEL1 and
homologs.
Supplemental Figure S7. Cell wall structure in wild-type and del1 roots
Supplemental Figure S8. Expression analysis of cell expansion-related
genes in culms.
Supplemental Table S1. The basic agronomic traits in wild-type and del1
plants.
Supplemental Table S2. Segregation analysis of F2 phenotype from
crosses 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 genes
identified 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 Developmental
Biology, Chinese Academy of Sciences) for help with the monosaccharide and
cellulose analyses, Dr. Yong Hu (Capital Normal University) for help with the
flow 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.
Plant Physiol. Vol. 174, 2017

LITERATURE CITED
Anderson CT, Carroll A, Akhmetova L, Somerville C (2010) Real-time
imaging of cellulose reorientation during cell wall expansion in Arabi-
dopsis roots. Plant Physiol 152: 787–796
Apel K, Hirt H (2004) Reactive oxygen species: Metabolism, oxidative
stress, and signal transduction. Annu Rev Plant Biol 55: 373–399
Atmodjo MA, Hao Z, Mohnen D (2013) Evolving views of pectin biosyn-
thesis. Annu Rev Plant Biol 64: 747–779
Barras F, Gijsegem FV, Chatterjee AK (1994) Extracellular enzymes and
pathogenesis of soft-rot Erwinia. Annu Rev Phytopathol 32: 201–234
Biswal AK, Soeno K, Gandla ML, Immerzeel P, Pattathil S, Lucenius J,
Serimaa R, Hahn MG, Moritz T, Jönsson LJ, et al (2014) Aspen pectate
lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues
and improves saccharification yield. Biotechnol Biofuels 7: 11
Blum A, Ebercon A (1981) Cell membrane stability as a measure of drought
and heat tolerance in wheat. Crop Sci 21: 43–47
Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative
determination of uronic acids. Anal Biochem 54: 484–489
Bonnin E, Garnier C, Ralet MC (2014) Pectin-modifying enzymes and
pectin-derived materials: Applications and impacts. Appl Microbiol
Biotechnol 98: 519–532
Bradford MM (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72: 248–254
Brummell DA (2006) Cell wall disassembly in ripening fruit. Funct Plant
Biol 33: 103–119
Collmer A, Ried JL, Mount MS (1988) Assay methods for pectic enzymes.
In WA Wood, ST Kellogg, eds, Methods in Enzymology. Academic
Press, San Diego, CA, pp 329–335
Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6:
850–861
Côté F, Ham KS, Hahn MG, Bergmann CW (1998) Oligosaccharide elici-
tors in host-pathogen interactions. Generation, perception, and signal
transduction. Subcell Biochem 29: 385–432
Dewitte W, Murray JAH (2003) The plant cell cycle. Annu Rev Plant Biol
54: 235–264
Dick-Pérez M, Zhang Y, Hayes J, Salazar A, Zabotina OA, Hong M (2011)
Structure and interactions of plant cell-wall polysaccharides by two- and
three-dimensional magic-angle-spinning solid-state NMR. Biochemistry
50: 989–1000
Domingo C, Roberts K, Stacey NJ, Connerton I, Ruíz-Teran F, McCann MC
(1998) A pectate lyase from Zinnia elegans is auxin inducible. Plant J 13: 17–28
Dominguez-Puigjaner E, LLop I, Vendrell M, Prat S (1997) A cDNA clone
highly expressed in ripe banana fruit shows homology to pectate lyases.
Plant Physiol 114: 1071–1076
Duan Y, Li S, Chen Z, Zheng L, Diao Z, Zhou Y, Lan T, Guan H, Pan R, Xue
Y, et al (2012) Dwarf and deformed flower 1, encoding an F-box protein, is
critical for vegetative and floral development in rice (Oryza sativa L.).
Plant J 72: 829–842
Fang C, Zhang H, Wan J, Wu Y, Li K, Jin C, Chen W, Wang S, Wang W, Zhang
H, et al (2016) Control of leaf senescence by an MeOH-Jasmonates cascade that
is epigenetically regulated by OsSRT1 in rice. Mol Plant 9: 1366–1378
Ferrari S, Savatin DV, Sicilia F, Gramegna G, Cervone F, Lorenzo GD
(2013) Oligogalacturonides: Plant damage-associated molecular patterns
and regulators of growth and development. Front Plant Sci 4: 49
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady
E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues.
Science 220: 1049–1051
Guo LB, Chu CC, Qian Q (2006) Rice mutants and functional genomics.
Chin Bull Bot 23: 1–13
Held MA, Be E, Zemelis S, Withers S, Wilkerson C, Brandizzi F (2011)
CGR3: A Golgi-localized protein influencing homogalacturonan meth-
ylesterification. Mol Plant 4: 832–844
Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of
rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of
the boundaries of the T-DNA. Plant J 6: 271–282
Hongo S, Sato K, Yokoyama R, Nishitani K (2012) Demethylesterification
of the primary wall by PECTIN METHYLESTERASE35 provides me-
chanical support to the Arabidopsis stem. Plant Cell 24: 2624–2634
Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative
analysis of large gene lists using DAVID bioinformatics resources. Nat
Protoc 4: 44–57
Plant Physiol. Vol. 174, 2017
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DEL1 Affects Rice Growth and Leaf Senescence
Huang L, Sun Q, Qin F, Li C, Zhao Y, Zhou DX (2007) Down-regulation of
a SILENT INFORMATION REGULATOR2-related histone deacetylase
gene, OsSRT1, induces DNA fragmentation and cell death in rice. Plant
Physiol 144: 1508–1519
Iwai H, Masaoka N, Ishii T, Satoh S (2002) A pectin glucuronyltransferase
gene is essential for intercellular attachment in the plant meristem. Proc
Natl Acad Sci USA 99: 16319–16324
Khanna-Chopra R (2012) Leaf senescence and abiotic stresses share reac-
tive oxygen species-mediated chloroplast degradation. Protoplasma 249:
469–481
Kim SH, Choi HS, Cho YC, Kim SR (2012) Cold-responsive regulation of a
flower-preferential class III peroxidase gene, OsPOX1, in rice (Oryza
sativa L.). J Plant Biol 55: 123–131
Koch E, Slusarenko A (1990) Arabidopsis is susceptible to infection by a
downy mildew fungus. Plant Cell 2: 437–445
Krizek BA (2009) Making bigger plants: Key regulators of final organ size.
Curr Opin Plant Biol 12: 17–22
Kulikauskas R, McCormick S (1997) Identification of the tobacco and
Arabidopsis homologues of the pollen-expressed LAT59 gene of tomato.
Plant Mol Biol 34: 809–814
Laskowski M, Biller S, Stanley K, Kajstura T, Prusty R (2006) Expression
profiling of auxin-treated Arabidopsis roots: Toward a molecular analysis
of lateral root emergence. Plant Cell Physiol 47: 788–792
Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58:
115–136
McQueen-Mason SJ, Cosgrove DJ (1995) Expansin mode of action on cell
walls. Analysis of wall hydrolysis, stress relaxation, and binding. Plant
Physiol 107: 87–100
Medina-Escobar N, Cárdenas J, Moyano E, Caballero JL, Muñoz-Blanco J
(1997) Cloning, molecular characterization and expression pattern of a
strawberry ripening-specific cDNA with sequence homology to pectate
lyase from higher plants. Plant Mol Biol 34: 867–877
Milioni D, Sado PE, Stacey NJ, Domingo C, Roberts K, McCann MC
(2001) Differential expression of cell-wall-related genes during the for-
mation of tracheary elements in the Zinnia mesophyll cell system. Plant
Mol Biol 47: 221–238
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen
species homeostasis and signalling during drought and salinity stresses.
Plant Cell Environ 33: 453–467
Nooden LD, Leopold AC (1978) Phytohormones and the endogenous
regulation of senescence and abscission. In DS Letham, PB Goodwin,
TJV Higgins, eds, Phytohormones and Related Compounds: A Compre-
hensive Treatise. Elsevier/North-Holland Biomedical Press, Amsterdam,
The Netherlands, 329–369.
Nunan KJ, Davies C, Robinson SP, Fincher GB (2001) Expression patterns
of cell wall-modifying enzymes during grape berry development. Planta
214: 257–264
Ogawa M, Kay P, Wilson S, Swain SM (2009) ARABIDOPSIS DEHISCENCE
ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 are
polygalacturonases required for cell separation during reproductive devel-
opment in Arabidopsis. Plant Cell 21: 216–233
Palusa SG, Golovkin M, Shin SB, Richardson DN, Reddy ASN (2007)
Organ-specific, developmental, hormonal and stress regulation of ex-
pression of putative pectate lyase genes in Arabidopsis. New Phytol 174:
537–550
Park YB, Cosgrove DJ (2012) A revised architecture of primary cell walls
based on biomechanical changes induced by substrate-specific endo-
glucanases. Plant Physiol 158: 1933–1943
Peaucelle A, Braybrook SA, Le Guillou L, Bron E, Kuhlemeier C, Höfte H
(2011) Pectin-induced changes in cell wall mechanics underlie organ
initiation in Arabidopsis. Curr Biol 21: 1720–1726
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0:
Discriminating signal peptides from transmembrane regions. Nat
Methods 8: 785–786
Pien S, Wyrzykowska J, McQueen-Mason S, Smart C, Fleming A (2001)
Local expression of expansin induces the entire process of leaf develop-
ment and modifies leaf shape. Proc Natl Acad Sci USA 98: 11812–11817
Pua EC, Ong CK, Liu P, Liu JZ (2001) Isolation and expression of two
pectate lyase genes during fruit ripening of banana (Musa acuminata).
Physiol Plant 113: 92–99
Ramirez-Parra E, Desvoyes B, Gutierrez C (2005) Balance between cell
division and differentiation during plant development. Int J Dev Biol 49:
467–477
1165
Leng et al.
Ren D, Rao Y, Leng Y, Li Z, Xu Q, Wu L, Qiu Z, Xue D, Zeng D, Hu J, et al
(2016) Regulatory role of OsMADS34 in the determination of glumes
fate, grain yield and quality in rice. Front Plant Sci 7: 1853
Ridley BL, O’Neill MA, Mohnen D (2001) Pectins: Structure, biosynthesis,
and oligogalacturonide-related signaling. Phytochemistry 57: 929–967
Rogers HJ, Harvey A, Lonsdale DM (1992) Isolation and characterization
of a tobacco gene with homology to pectate lyase which is specifically
expressed during microsporogenesis. Plant Mol Biol 20: 493–502
Romualdi C, Bortoluzzi S, D’Alessi F, Danieli GA (2003) IDEG6: A web
tool for detection of differentially expressed genes in multiple tag
sampling experiments. Physiol Genomics 12: 159–162
Sakuraba Y, Rahman ML, Cho SH, Kim YS, Koh HJ, Yoo SC, Paek NC
(2013) The rice faded green leaf locus encodes protochlorophyllide oxi-
doreductase B and is essential for chlorophyll synthesis under high light
conditions. Plant J 74: 122–133
Simeonova E, Sikora A, Charzy nska M, Mostowska A (2000) Aspects of
programmed cell death during leaf senescence of mono- and dicotyle-
donous plants. Protoplasma 214: 93–101
Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J,
Osborne E, Paredez A, Persson S, Raab T, et al (2004) Toward a sys-
tems approach to understanding plant cell walls. Science 306: 2206–2211
Song XQ, Liu LF, Jiang YJ, Zhang BC, Gao YP, Liu XL, Lin QS, Ling HQ,
Zhou YH (2013) Disruption of secondary wall cellulose biosynthesis alters
cadmium translocation and tolerance in rice plants. Mol Plant 6: 768–780
Sun L, van Nocker S (2010) Analysis of promoter activity of members of the
PECTATE LYASE-LIKE (PLL) gene family in cell separation in Arabi-
dopsis. BMC Plant Biol 10: 152
Tan L, Eberhard S, Pattathil S, Warder C, Glushka J, Yuan C, Hao Z, Zhu
X, Avci U, Miller JS, et al (2013) An Arabidopsis cell wall proteoglycan
consists of pectin and arabinoxylan covalently linked to an arabinoga-
lactan protein. Plant Cell 25: 270–287
Tan J, Tan Z, Wu F, Sheng P, Heng Y, Wang X, Ren Y, Wang J, Guo X,
Zhang X, et al (2014) A novel chloroplast-localized pentatricopeptide
repeat protein involved in splicing affects chloroplast development and
abiotic stress response in rice. Mol Plant 7: 1329–1349
Updegraff DM (1969) Semimicro determination of cellulose in biological
materials. Anal Biochem 32: 420–424
Van Sandt VS, Suslov D, Verbelen JP, Vissenberg K (2007) Xyloglucan
endotransglucosylase activity loosens a plant cell wall. Ann Bot (Lond)
100: 1467–1473
Verhertbruggen Y, Marcus SE, Haeger A, Ordaz-Ortiz JJ, Knox JP (2009)
An extended set of monoclonal antibodies to pectic homogalacturonan.
Carbohydr Res 344: 1858–1862
Vincken JP, Schols HA, Oomen RJFJ, McCann MC, Ulvskov P, Voragen AGJ,
Visser RGF (2003) If homogalacturonan were a side chain of rhamnoga-
lacturonan I. Implications for cell wall architecture. Plant Physiol 132: 1781–1789
Vogel JP, Raab TK, Schiff C, Somerville SC (2002) PMR6, a pectate lyase-
like gene required for powdery mildew susceptibility in Arabidopsis.
Plant Cell 14: 2095–2106
Wang X, Fang G, Li Y, Ding M, Gong HY, Li YS (2013) Differential anti-
oxidant responses to cold stress in cell suspension cultures of two sub-
species of rice. Plant Cell Tissue Organ Cult 113: 353–361
Wang H, Guo Y, Lv F, Zhu H, Wu S, Jiang Y, Li F, Zhou B, Guo W, Zhang
T (2010) The essential role of GhPEL gene, encoding a pectate lyase, in
1166
Downloaded from on August 19, 2019 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
cell wall loosening by depolymerization of the de-esterified pectin
during fiber elongation in cotton. Plant Mol Biol 72: 397–406
Wang Z, Wang Y, Hong X, Hu D, Liu C, Yang J, Li Y, Huang Y, Feng Y,
Gong H, et al (2015) Functional inactivation of UDP-N-acetylgluco-
samine pyrophosphorylase 1 (UAP1) induces early leaf senescence and
defence responses in rice. J Exp Bot 66: 973–987
Willats WGT, McCartney L, Knox JP (2001) In-situ analysis of pectic
polysaccharides in seed mucilage and at the root surface of Arabidopsis
thaliana. Planta 213: 37–44
Wing RA, Yamaguchi J, Larabell SK, Ursin VM, McCormick S (1990)
Molecular and genetic characterization of two pollen-expressed genes
that have sequence similarity to pectate lyases of the plant pathogen
Erwinia. Plant Mol Biol 14: 17–28
Wolf S, Hématy K, Höfte H (2012) Growth control and cell wall signaling
in plants. Annu Rev Plant Biol 63: 381–407
Wolf S, Mouille G, Pelloux J (2009) Homogalacturonan methyl-esterification
and plant development. Mol Plant 2: 851–860
Wu Y, Qiu X, Du S, Erickson L (1996) PO149, a new member of pollen
pectate lyase-like gene family from alfalfa. Plant Mol Biol 32: 1037–1042
Wu HB, Wang B, Chen Y, Liu YG, Chen L (2013) Characterization and fine
mapping of the rice premature senescence mutant ospse1. Theor Appl
Genet 126: 1897–1907
Xiao C, Somerville C, Anderson CT (2014) POLYGALACTURONASE
INVOLVED IN EXPANSION1 functions in cell elongation and flower
development in Arabidopsis. Plant Cell 26: 1018–1035
Yadav S, Yadav PK, Yadav D, Yadav KDS (2009) Pectin lyase: A review.
Process Biochem 44: 1–10
Yang JL, Li YY, Zhang YJ, Zhang SS, Wu YR, Wu P, Zheng SJ (2008) Cell
wall polysaccharides are specifically involved in the exclusion of alu-
minum from the rice root apex. Plant Physiol 146: 602–611
Yang Y, Xu J, Huang L, Leng Y, Dai L, Rao Y, Chen L, Wang Y, Tu Z, Hu J,
et al (2016) PGL, encoding chlorophyllide a oxygenase 1, impacts leaf
senescence and indirectly affects grain yield and quality in rice. J Exp
Bot 67: 1297–1310
Yu L, Chen X, Wang Z, Wang S, Wang Y, Zhu Q, Li S, Xiang C (2013)
Arabidopsis enhanced drought tolerance1/HOMEODOMAIN GLABROUS11
confers drought tolerance in transgenic rice without yield penalty. Plant
Physiol 162: 1378–1391
Zhang SJ, Song XQ, Yu BS, Zhang BC, Sun CQ, Knox JP, Zhou YH (2012)
Identification of quantitative trait loci affecting hemicellulose charac-
teristics based on cell wall composition in a wild and cultivated rice
species. Mol Plant 5: 162–175
Zhang M, Zhang B, Qian Q, Yu Y, Li R, Zhang J, Liu X, Zeng D, Li J, Zhou
Y (2010) Brittle Culm 12, a dual-targeting kinesin-4 protein, controls cell-
cycle progression and wall properties in rice. Plant J 63: 312–328
Zhong H, Lauchli A (1993) Changes of cell wall composition and polymer
size in primary roots of cotton seedlings under high salinity. J Exp Bot
44: 773–778
Zhou B, Guo Z (2009) Calcium is involved in the abscisic acid-induced
ascorbate peroxidase, superoxide dismutase and chilling resistance in
Stylosanthes guianensis. Biol Plant 53: 63–68
Zou HY, Wenwen YH, Zang GC, Kang ZH, Zhang ZY, Huang JL, Wang
GX (2015) OsEXPB2, a b-expansin gene, is involved in rice root system
architecture. Mol Breed 35: 41
Plant Physiol. Vol. 174, 2017