Materials and Methods
Constructions for the complementation test,
tissue-specific expression, and subcellular localization of OsCASP1
protein
Plasmids were constructed using the In-Fusion cloning kit (Takara). To
generate OsCASP1pro:OsCASP1 , 1128 bp upstream of theOsCASP1 gene, including the whole intergenic sequence between
Os04g0684200 and Os04g0684200 (OsCASP1 ) and the OsCASP1gene were amplified, and the PCR product was recombined into
pCAMBIA-1300 digested with Hind III with the In-Fusion cloning kit. To
generate OsCASP1pro:OsCASP1-GUS andOsCASP1pro:OsCASP1-GFP , the promoter (including whole intergenic
sequence) and OsCASP1 gene were amplified and recombined into
pCXGUS-P and pCXGFP-P digested with Xcm I, respectively. The constructs
of 35S::DsRed-OsCASP1, 35S::DsRed-OsCASP1-N, and35S::DsRed-OsCASP1-C were generated by recombining theOsCASP1 gene into pCX-DR. The details of these constructs are
shown in figure S3, and the primers used in this study were shown in
table S4. The markers for the nucleus and endoplasmic reticulum were
used for transient expression for the subcellular localization of
OsCASP1 (Z. Chen et al., 2019).
For the complementation experiment, OsCASP1pro:OsCASP1 was
transformed into a few F3 progenies of the els1mutant and Nipponbare, which exhibited fewer tillers and leaf cell death
and looked like Nipponbare with respect to morphological phenotype. The
constructs of OsCASP1pro:OsCASP1-GUS ,OsCASP1pro:OsCASP1-GFP, and 35S::DsRed-OsCASP1 were
transformed into Zhonghua 11. The CRISPR/cas9 mutant of OsCASP1were generated by the Biogle Company, and the target sequence was
localized to exon 1 of the OsCASP1 gene.
Growth Conditions
Rice plants were grown under two different conditions: soil and
hydroponics. For soil experiments, the F2 and
F3 populations of the cross of the els1 mutant
and Nipponbare were grown in the experimental paddy field in Putian,
Fujian province. Rice plants were grown in a growth cabinet under
28°C/14-h light and 28°C/10-h dark photoperiod in nutrient solution
(Table S2) to test the sensitivity of the plants to nutrient deficiency.
DNA and Protein Sequence
Analysis
The new molecular markers were designed according to the Nipponbare
sequence, and the CASP and CASP-like proteins in rice were obtained by
searching the RAP-DB database with the BlastP programmes. The Molecular
Evolutionary Genetic Analysis programme (MEGA X) was used to generate
phylogenetic trees using the maximum likelihood method and JTT
matrix-based model (Kumar, Stecher, Li,
Knyaz, & Tamura, 2018).
Histochemical staining
Roots of 10~14-d-old seedling growing in nutrient
solution were used for the study, and freehand cross-sections were cut
at different regions. To observe the Casparian strip, root
cross-sections were stained with 0.1% (w/v) berberine chloride and
0.5% (w/v) aniline blue as described by Brundrett et al.
(1988)(Brundrett, Enstone, & Peterson,
1988). Casparian strips were visualized as bright white/yellow
fluorescence (UV filter set). To visualize lignin with cinnamyl aldehyde
groups in the roots, cross-sections were stained for 30 min with 1%
phloroglucinol in 20% (w/v) hydrochloric acid at room temperature.
Lignin appears orange/red under white light
(Pradhan Mitra & Loque, 2014;
Shiono et al., 2014). For the periodic
acid staining, root cross-sections were incubated in 0.1% (w/v)
H5IO6 for 60 min and washed with
reducing solution (1 g of KI and 1 g of
Na2S2O3.5H2O
dissolved in 50 ml of H2O and acidified with 0.5 ml of 2
M HCl) for 60 min. The cross-sections were visualized as purple staining
in the cell walls under a light microscope
(Shiono et al., 2014). Suberin deposition
in the roots was visualized with Fluorol Yellow 088 as described by
Barberon et al.(Barberon et al., 2016).
Electron Microscopy
The state I endodermis was visualized using root sections collected
5~10 mm from the root tips in 17-d-old seedlings.
Transmission electron microscopy (TEM) was conducted using a previously
described protocol (Hulskamp, Schwab,
Grini, & Schwarz, 2010).
Permeability test
For the propidium iodide penetration assay, the roots of
8~13-d-old seedlings were incubated in the dark for 40
min in a fresh solution of 15 μM (10 μg/ml) propidium iodide and rinsed
two times in water. They were then visualized with a confocal
laser-scanning microscope(Naseer et al.,
2012). The permeability of the exodermal and endodermal layers of roots
was assessed with periodic acid and berberine as described by Shiono et
al.(Shiono et al., 2014)