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Pcy 175
Pcy 175
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2
College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
*Corresponding author: E-mail, [email protected]; Fax, +86-691-8715070.
Subject area: Growth and development
(Received May 11, 2018; Accepted August 21, 2018)
Jatropha curcas is a promising feedstock for biofuel production have gained significant attention as a promising fuel (Mofijur
Plant Cell Physiol. 59(12): 2549–2563 (2018) doi:10.1093/pcp/pcy175, Advance Access publication on 24 August 2018,
available online at www.pcp.oxfordjournals.org
! The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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M. Tang et al. | miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas
expressed miRNAs, lineage (lin)-4 and lethal (let)-7 (Pasquinelli soybean (Yan et al. 2013). Also miR172 was found to have a
and Ruvkun 2002, Carrington and Ambros 2003, Moss 2007). role in the abiotic response of Arabidopsis (Han et al. 2013) and
lin-4 and let-7 were the first miRNAs identified, and they have biotic stress resistance in tomato (Luan et al. 2018). In addition,
since served as paradigms for the functions of these regulatory miR172 causes early flowering and defects in floral organ iden-
molecules in animals (Bagga et al. 2005). MicroRNAs function as tity when overexpressed (Aukerman and Sakai 2003).
post-transcriptional modulators of gene expression in eukaryotic Furthermore, miR172 promotes flowering primarily by post-
cells (Inui et al. 2010). In the annual plants Arabidopsis and maize, transcriptionally repressing a set of AP2-like genes, such as
vegetative phase change was controlled by the sequential activity AP2, TOE1, TOE2, TOE3, SMZ, and SNZ (Fornara and
of miR156 and miR172 (Chuck et al. 2007a, Wu et al. 2009). Coupland 2009, Mathieu et al. 2009, Yant et al. 2010).
miR156 is highly expressed early in plant development and de- Previous studies have shown that miR172-overexpressing
creases with time, while miR172 exhibits the opposite expression Arabidopsis plants exhibit early flowering by regulating
pattern (Aukerman and Sakai 2003, Lauter et al. 2005, Jung et al. FLOWERING LOCUS T (FT) (Lee et al. 2007, Lee et al. 2010,
2007, Wang et al. 2009, Wu et al. 2009). In woody species, such as Diaz-Manzano et al. 2018). FRUITFULL (FUL) positively regu-
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miR172 target sites are contained in the coding sequences and Jatropha. Similar miR172 expression patterns had also been
30 -UTR regions of the target genes (Fig. 1A). observed in Arabidopsis, rice and maize (Aukerman and Sakai
To determine where miR172 is expressed during Jatropha 2003, Lauter et al. 2005, Zhu et al. 2009).
development, we analyzed the miR172 expression profiles in
various tissues by qRT-PCR according to Varkonyi-Gasic et al. Characterisation of the JcmiR172a functions in
(2007). The mature miR172a–e sequences differ only in their 50 transgenic Arabidopsis
and 30 end bases (Supplementary Fig. S1), and therefore, a pair of To test the functions of JcmiR172a, we transformed the 35S:
miR172a primers can detect the expression of all mature JcmiR172a construct into Arabidopsis for preliminary analysis.
miR172s; qRT-PCR was performed with total RNAs extracted Transgenic plants were confirmed by qRT-PCR analysis the
from various tissues. The results indicated that there was an expression level of miR172 (Supplementary Fig. S2). We selected
age-related increase in miR172 expression in Jatropha (Fig. 1B), two independent homozygous lines, L2 and L4, in the T2 gener-
which is consistent with a recent study of dynamics of miR172s ation to further examine the phenotypes. L2 and L4 generated
during a production cycle of Jatropha (Sánchez-Gutiérrez et al. flowers 14–15 days earlier and produced approximately nine
2018). The lowest expression level was observed in the first leaf fewer rosette leaves than that of WT plants under LD conditions
of the seedlings rather than cotyledons; with the age increasing, (Fig. 2A–C; Table 1). Therefore, overexpression of JcmiR172a in
the transcription level of miR172 was increased continually. The Arabidopsis significantly reduced vegetative growth time.
highest level was observed in five-year-old plants (Fig. 1B). The Furthermore, compared with WT plants, the transgenic
expression pattern of miR172 in Jatropha plants of different ages plants showed altered leaf sizes and morphologies (Fig. 2D
was consistent with that in other trees (Wang et al. 2011). We and E). The sizes of rosette and cauline leaves were reduced
also examined the miR172 expression levels in different organs of in transgenic Arabidopsis. In contrast to the rosette leaves of
Jatropha plants. As shown in Fig. 1C, miR172 was expressed in all WT, which exhibited obvious serrations, the rosette leaves of
organs. The highest level was exhibited in the leaves, and high transgenic plants showed a smooth edge. Furthermore, the leaf
expression was observed in the shoot apex; miR172 expression basal angle was smaller than that of the WT plants (Fig. 2D–G),
varied considerably between organs and developmental stages of which is similar to the results reported by Jung et al. (2011).
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M. Tang et al. | miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas
Table 1 Overexpression of JcmiR172a promotes flowering in We further analyzed the gene expression in 35S: JcmiR172a
Arabidopsis under LD conditions transgenic Arabidopsis plants showing early flowering and ab-
Lines Number of Rosette leaves Flower bud normal flowers via qRT-PCR. The results showed that high
plants formation time/Day miR172 levels down-regulated several AP2-like genes, including
WT 25 12.21 ± 1.04 24.56 ± 1.33 AtAP2, AtTOE1, AtTOE2, AtSMZ, and AtSNZ, in seedlings and
L2 35 3.56 ± 0.75** 9.47 ± 1.44** flowers (Supplementary Fig. S2), which act as flowering repres-
L4 37 3.92 ± 0.63** 10.13 ± 1.38** sors (Aukerman and Sakai 2003). In addition, the expression of
WT plants and two independent JcmiR172a-overexpressing lines (L2 and L4)
the floral meristem identity genes AtLEAFY (AtLFY), AtFUL,
grown under LD conditions (16 h light/8 h dark) were subjected to the analysis AtAP1 and AtCAL and the floral organ identity genes AtSEPs
of rosette leaves and flowering times. The rosette leaves and flowering times are (AtSEP1, AtSEP2, AtSEP3) were significantly up-regulated in ten-
presented as the mean ± standard deviation. **Significantly different from the
control at the 1% level.
day-old transgenic seedlings (Supplementary Fig. S2). However,
the floral organ identity genes AtAGAMOUS (AtAG), AtAP3 and
AtSEP2 were down-regulated in transgenic Arabidopsis flowers
The fourth rosette leaf of the 35S: JcmiR172a transgenic plants (Supplementary Fig. S2).
was ovate with few trichomes on the adaxial side (Fig. 2G, The phenotypes of early flowering and abnormal flowers
Supplementary Fig. S3), which represents a leaf morphology produced by ectopic expression of JcmiR172a in transgenic
associated with the adult vegetative phase (Wu et al. 2009). Arabidopsis were similar to those produced by miR172 over-
More trichomes on the abaxial side, however, were found in expression (Chen 2004). Gene expression analysis indicated
transgenic Arabidopsis than that in WT plants (Supplementary that miR172 caused early flowering by down-regulating AP2-
Fig. S3). These observations indicate that miR172 also regulates like floral repressors. The abnormal flowers appeared due to
developmental timing in addition to floral induction. miR172 directly repressing the floral identity gene AP2 and in-
The floral pattern was also changed in transgenic directly repressing other floral identity genes, including AtAP3,
Arabidopsis. The sepals, petals, and pistils were all smaller AtAG, and AtSEP2 (Supplementary Fig. S2).
than that in WT (Fig. 3B–G and J); the petals of each flower
were variable in size (Fig. 3G). Petals and stamens were partially Overexpression of JcmiR172a in Jatropha changed
absent (Fig. 3C, I), and the absent stamens in the third the leaf and stem morphologies
whorl were fused to the petals in the second whorl in transgenic We generated transgenic Jatropha plants overexpressing the
Arabidopsis (Fig. 3G). The major phenotypes of JcmiR172a- JcmiR172a precursor driven by the 35S promoter. Elevated
overexpressing plants were similar to those of the Arabidopsis levels of miR172 were detected in JcmiR172a overexpression
ap2 mutants described by Kunst et al. (1989). plants (Supplementary Fig. S4A). The leaves of the transgenic
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Jatropha were smaller, the leaf shape and leaf margin were diameter of miR172-overexpressing Arabidopsis was reduced
altered, and the petiole length, leaf length and width were by approximately 0.4–0.6 mm (Supplementary Fig. S5D); the
all significantly reduced compared to those of the WT results of transgenic Arabidopsis stem were similar to those
plants (Fig. 4A, B, Table 2). The leaf lobes disappeared in of transgenic Jatropha. A comparison of the stem structure
mature leaves of transgenic Jatropha plants (Fig. 4A, B). The from pictures of shoot cross-sections of stems from different
stem morphologies also changed in transgenic Jatropha. We genotype plants showed that the number of vascular bundles
compared 20 progeny seedlings of L32 and L47 transgenic was reduced in transgenic Arabidopsis. Generally, there are
plants with WT plants and found that the transgenic progeny eight vascular bundles in WT Arabidopsis, but there were
seedings exhibited a thinner but harder stem phenotype only 5 or 6 vascular bundles in 35S: JcmiR172a transgenic
(Fig. 4C–F). A comparison of stems of miR172 transgenic plants (Fig. 5A–C). To further examine xylem-related
Jatropha and WT via cross-sections and longitudinal sections traits, we prepared paraffin cross-sections of basal stems, and
revealed that the thickness of transgenic Jatropha xylem was different vascular bundle attributes, including area and number
0.36–0.75 mm thicker than that of WT in two-month-old of cell rows and lines, were evaluated (Fig. 5D, Supplementary
seedlings (Fig. 4C, E) and 1.5 mm thicker in five-month-old Fig. S5). Both numbers of xylem cell rows and lines were signifi-
seedlings (Fig. 4D). The percentage of xylems of transgenic cantly higher in 35S: JcmiR172a transgenic plants than WT
Jatropha increased by approximately 10% compared to that of plants (Supplementary Fig. S5E, F). Furthermore, we recorded
WT, whereas the percentages of phloem and piths were sig- the ratio of the xylem/stem area calculated as the fraction of
nificantly reduced (Fig. 4C–F). total xylem area with respect to the total stem area ratio. This
parameter reflected the proportion of xylem in the total stem
Overexpression of JcmiR172a enhanced xylem
area. Notably, we observed a significant increase in the xylem
development via regulation of cell size and
ratio among the 35S: JcmiR172a transgenic plants and WT
number in vascular tissues of Arabidopsis and plants (Fig. 5D).
Jatropha The increased xylem may result in an increase of xylem cell
In this study, we first found the miR172 regulates xylem devel- volume. To verify this hypothesis, we analyzed the paraf-
opment. To confirm this function of miR172, we further sur- fin cross-sections with an oil immersion lens. Unexpectedly, a
veyed the stem traits in JcmiR172a transgenic Arabidopsis using comparison of the xylem cell morphologies showed that the
40-day-old T2 seedings. Notably, we also found that the stem xylem cell size in 35S: JcmiR172a plants was smaller than that in
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M. Tang et al. | miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas
Table 2 Characteristics of mature leaves and flowering time of JcmiR172a transgenic plants
Sample N Petiole length (cm) Leaf length (cm) Leaf width (cm) Flower bud formation
time/Day
T0 WT 24 17.67. ± 1.65c 15.00 ± 1.05c 16.07 ± 1.71b -
L32 19 12.72 ± 1.13b 12.07 ± 1.07b 11.13 ± 1.00a -
L47 21 10.96 ± 1.42a 10.93 ± 0.92a 10.47 ± 0.99a -
T1 WT 15 6.95 ± 1.58c 10.42 ± 0.95c 11.53 ± 0.81c 324.56 ± 53.61B
L32 15 5.47 ± 0.74b 8.17 ± 0.85b 8.97 ± 0.91b 139.47 ± 15.42A
L47 15 4.48 ± 0.43a 6.49 ± 0.73a 7.15 ± 0.51a 120.13 ± 13.24A
Leaves of WT plants and two independent JcmiR172a-overexpressing lines (L32 and L47) grown in the field were subjected to an analysis of leaf size. N = sample
number. The petiole length, leaf length, leaf width, and flower bud formation time are presented as the mean ± standard deviation. Values with different lowercase
letters are significantly different at P < 0.05 by Tukey’s test. Values with different capital letters are significantly different at P < 0.01 by Tukey’s test.
WT plants (Fig. 6). However, the xylem cell density was higher We further analyzed the lignification-related genes in 35S:
than that in WT. Analysis of the microphotographs showed JcmiR172a transgenic Jatropha via qRT-PCR. Total RNA samples
that in WT plants there were only 150 xylem cells in a were extracted from stems of two-month-old T1 transgenic
200 mm 200 mm zone, but in miR172 overexpression plants, and WT seedings. The results showed that the expression
there were 220–230 xylem cells in a 200 mm 200 mm zone levels of the lignin biosynthesis genes Cinnamyl alcohol de-
(Fig. 6D–F). In transgenic Arabidopsis, the xylem cell size was hydrogenase 6 (CAD6) and caffeoyl CoA O-methyltransferase
also smaller than that in WT (Fig. 5E–G), and the smallest cell (CCoAOMT) were strongly up-regulated (Fig. 7A, B), and the
size was observed in plant line 2, which had the highest miR172 cellulose synthase A (CesA) genes JcCesA1, JcCesA3, JcCesA4,
expression level (Supplementary Fig. S2A). These results were JcCesA7, and JcCesA8 were also substantially up-regulated
consistent with the findings in transgenic Jatropha. The (Fig. 7C, F, H–J). However, the expression levels of JcCesA2,
changes in xylem traits were mostly explained by an increased JcCesA2-L, and JcCesA3-L showed no significant increases
number of xylem cell rows, which caused increased total xylem (Fig. 7D, E, G). These results indicated that JcmiR172 acceler-
thickness and ratio (Figs. 5, 6, Supplementary Fig. S5). ates transgenic Jatropha xylem development by indirectly
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Fig. 6 Comparison of the xylem cell morphology and density between WT and 35S: JcmiR172a transgenic Jatropha. (A–C) Paraffin sections of
WT (A), 35S: JcmiR172a L32 (B) and L47 (C), 200 mm 200 mm zones are marked by red squares. (D–F) Magnifying marked zone of (A–C)
respectively, the number of cells in this zone were counted; bar = 100 mm.
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M. Tang et al. | miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas
promoting the expression of lignin biosynthesis and cellulose that the transcript levels of the miR172 target genes JcAP2,
synthase genes. JcTOE1, and JcTOE2 were down-regulated (Supplementary Fig.
S4B–D), and the transcript levels of JcFT, JcLFY, and JcAP1 were
Overexpression of JcmiR172a in Jatropha caused slightly altered in the transgenic seedlings (Supplementary Fig.
early flowering and abnormal development of S4F–H). However, the expression levels of JcSOC1 and JcSEP2
reproductive organs were increased more than 10-fold (Supplementary Fig. S4I, M).
Transgenic analysis performed in Arabidopsis suggested that These results indicated that miR172 is involved in floral meri-
miR172 might act as a flowering accelerator in Jatropha. To stem determination in Jatropha.
test this hypothesis, we generated transgenic Jatropha with Comparing the flowers, we found that the floral organ patterns
the 35S: JcmiR172a construct as previously described. Non- were obviously different (Fig. 8A, B). We dissected the flowers and
transgenic/WT plants were used as control. Fifty independent found that the flower organs were partially absent in transgenic
transgenic lines were confirmed via PCR. To our surprise, all plants; in the first and second whorls, there were 2–4 sepals and 2–
the transgenic Jatropha lines lacked the early-flowering pheno- 3 petals. In addition, 2–3 nectaries were observed. In WT flowers,
type in a tropical area (Xishuangbanna). When regenerated there were five sepals, petals and nectaries (Fig. 8A–F; Table 3). In
plantlets (Supplementary Fig. S6) were grown in the field for male flowers, 5–6 stamens were observed, while WT flowers had 10
5 months, flower buds emerged in both transgenic and control stamens (Fig. 8G, H; Table 3), and similar to transgenic Arabidopsis
plants (Supplementary Fig. S6D–G). In contrast, two (L8 and (Fig. 3G), there were two stamens fused to the petals (Fig. 8F). The
L21) of the 15 transgenic lines of the 35S: JcmiR172a-overex- carpels were present, but the ovules are partially absent in female
pressing Jatropha grown in a subtropical area (Kunming) pro- flowers (Fig. 8I, J; Table 3). The expression profiles showed that the
duced flowers in the second year (Supplementary Fig. S7B–C, transcript levels of floral organ identity genes, including JcAP2,
E–F). The transgenic plants produced flowers only once and JcAP3, JcAG, JcSEP1, and JcSEP3 (Supplementary Fig. S4B and J–L,
only in spring every year, while the WT plants did not produce N), were down-regulated in transgenic flowers. Because the abun-
flowers until 5 years after they were planted in the same con- dance of miR172 in wild-type plants is low in young seedlings and
ditions (Supplementary Fig. S7A, D). We further analyzed the T1 high in inflorescences, the increase in miR172 abundance in the 35S:
seedling phenotypes and several floral identity-related genes in JcmiR172a lines was more evident in young seedlings than in in-
a greenhouse in a tropical area. We found that the T1 trans- florescences. In addition, in 35S: JcmiR172a transgenic plants, the
genic plants generated flower buds at least 5 to 6 months earlier miR172 levels were not significantly different at different ages
than the WT plants (Table 2). The expression results showed (Supplementary Fig. S4A).
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The results obtained from the transgenic Jatropha indicate bigger, but the weight and oil contents of these seeds were
that miR172 is important in regulating flower organ develop- decreased compared to those of WT seeds (Fig. 9E, F,
ment and sustaining normal patterns through regulation of the Table 4). The changes in size, number, weight, and oil contents
expression of floral organ identity genes. of seeds in JcmiR172a-overexpressing transgenic plants suggest
Jatropha plants overexpressing JcmiR172a showed signifi- that miR172 may be involved in seed development.
cant floral organ defects, altered fruit shape, and reduced
seed yield compared to WT plants. In transgenic plants, the
length of the fruits was longer than that of WT plants, Discussion
the width of the fruits was narrower than that of WT, and
the ratio of length to width (L/W) was increased (Fig. 9A, B).
Functional conservation and divergence of
Further analysis of the transgenic fruits showed that some seeds miR172 in Arabidopsis and Jatropha
in the fruits were aborted (Fig. 9C). One or two abortive seeds There are five pri-miR172s in both Arabidopsis and Jatropha.
were observed in each fruit; on average, the normal seed The sequences of the mature JcmiR172a-d and JcmiR172e,
number in each fruit was 1.31.6 (Fig. 9D), but in WT, the which have been isolated in Jatropha seeds (Galli et al. 2014),
seed number of each fruit was three, indicating a significant differ only in their 50 and 30 end bases (Supplementary Fig. S1A).
reduction in transgenic fruits. We compared the transgenic JcmiR172e, which is different from that of Arabidopsis miR172,
seeds to WT seeds and found that the transgenic seeds were shows higher similarity to that of some woody plants, such as
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M. Tang et al. | miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas
apple (Malus domestica), P. trichocarpa, Vitis vinifera, R. com- showed an increase in xylem thickness (Fig. 4C–F); further-
munis, and Citrus sinensis (http://www.mirbase.org/). more, transgenic Jatropha exhibited early flowering in a sub-
According to the alignment of nucleotide sequences, we tropical area (Supplementary Fig. S7). These results indicate
found JcmiR172e showed higher matching scores with tar- miR172 is an age marker gene in Jatropha, which is similar to
get genes than that of JcmiR172a-d (Fig.1A, Supplementary its role in other plants (Wang et al. 2011, Zhu and Helliwell
Fig. S1B). And the apple Md-miR172e, a homolog of 2011, Lee et al. 2014). In transgenic Arabidopsis and Jatropha,
JcmiR172e, has been shown to alter flowering time and floral the leaf morphologies were changed, the leaves were smaller,
organ identity when ectopically expressed in Arabidopsis (Zhao and the leaf margins were smoother than those in WT (Fig. 2E,
et al. 2015). F; and Fig. 4A, B). The flower organs were partially defective in
In this study, we showed the results of JcmiR172a overex- transgenic Arabidopsis and Jatropha (Figs. 3, 8). Comparing the
pression in both Arabidopsis and Jatropha. In Arabidopsis, results in these two species, and together with characterization
miR172 overexpression plants showed extremely early flower- of miR172 in other non-model plants (Glazińska et al. 2009, Nair
ing (Fig. 2). This result indicates that miR172 promotes the et al. 2010, Debernardi et al. 2017, Anwar et al. 2018, Shivaraj
transition from the vegetative phase to the adult phase. In et al. 2018), we concluded that the miR172 has conservative
Jatropha, JcmiR172a expression level continuously increased functions in regulating phase change, controlling leaf morphol-
with age (Fig. 1B), and JcmiR172a overexpression plants ogies, and sustaining normal flower development.
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Plant Cell Physiol. 59(12): 2549–2563 (2018) doi:10.1093/pcp/pcy175
In Arabidopsis three mature miR172s and six target genes, produce flowers continuously and did not produce flowers at
including AP2, SMZ, SNZ, TOE1, TOE2, and TOE3, were identified other seasons. The results indicated that miR172 cannot pro-
(Fornara and Coupland 2009, Mathieu et al. 2009). However, mote flowering independently; it also depends on suitable en-
there are only two mature miR172s in Jatropha (Supplementary vironmental conditions.
Fig. S1) and only four target genes of miR172, including JcAP2, Similar to our findings, overexpression of the rice miR172b in
JcTOE1, JcTOE2, and JcTOE3 (Fig. 1). Different target gene num- rice did not lead to early flowering (Zhu et al. 2009). Recent
bers may lead to divergent functions. studies in Cardamine flexuosa and Arabis alpine demonstrated
In transgenic Arabidopsis, the flower bud appeared that age is necessary but not sufficient to promote flowering
14–15 days earlier than that of WT, and it produced only 3–4 in perennial herbaceous plants, and other factors, such as low
rosette leaves. This phenotype was as strong as that shown by temperature, are also necessary (Bergonzi et al. 2013, Zhou et al.
Aukerman and Sakai (2003) and Lee et al. (2010). However, we 2013). Jatropha is a perennial woody plant. The molecular mech-
did not obtain transgenic Arabidopsis plants that completely anisms controlling flowering in perennial woody plants have not
lacked flower organs, but the absent flower organ phenotypes been studied as extensively as those of herbaceous plants (Albani
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M. Tang et al. | miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas
wood-related genes (Wong et al. 2011). However, a functional restriction enzymes KpnI and SalI and then cloned into the pOCA30 vector
containing the CaMV 35S promoter, resulting in the binary vector pMYT34.
analysis of these putative miRNAs with potential roles in wood
Transformation of WT Arabidopsis with Agrobacterium strain EHA105 carrying
formation has not been carried out. In this study, we demon- the pMYT34 (35S: JcmiR172a) was performed using the floral dip method
strated the function of miR172 in regulating wood formation in (Clough and Bent 1998). Transformation of Jatropha with Agrobacterium
Jatropha and Arabidopsis. strain EHA105 carrying the same construct was performed according to the
Several factors, such as the plant hormones gibberellin protocol described by Pan et al. (2010) and Fu et al. (2015). All transgenic plants
(Wang et al. 2017), auxin (Moreno-Piovano et al. 2017), jasmo- were confirmed by genomic PCR and RT-PCR.
nic acid and cytokinin (Jang et al. 2017), and some transcription qRT-PCR analysis
factors, e.g. bHLH complexes (Ohashi-Ito and Fukuda 2016),
Jatropha total RNA was extracted from frozen tissues according to the methods
LAX2 (Moreno-Piovano et al. 2017), VND6, VND7, NST3, and
described by Ding et al. (2008). Arabidopsis total RNA was extracted from
WOX4 (Stein et al. 2016), are involved in these processes. frozen tissues using TRIzol reagent (Transgene, China). First-strand comple-
However, the secondary xylem thickening phenotype was mentary DNA (cDNA) was synthesized from 1 mg of total RNA using the
never reported in miR172-overexpressing plants because the PrimeScriptÕ RT Reagent Kit (TaKaRa, Dalian, China) according to the instruc-
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Plant Cell Physiol. 59(12): 2549–2563 (2018) doi:10.1093/pcp/pcy175
Divakara, B., Upadhyaya, H., Wani, S. and Gowda, C. (2010) Biology and
Author contributions
genetic improvement of Jatropha curcas L.: a review. Appl. Energy 87:
Mingyong Tang designed and performed the experiments, ana- 732–742.
lyzed the data, and wrote the paper. Xue Bai, analyzed data and Fornara, F. and Coupland, G. (2009) Plant phase transitions make a SPLash.
Cell 138: 625–627.
revised the paper. Long-Jian Niu, Xia Chai, and Mao-Sheng Chen
Fouracre, J.P. and Poethig, R.S. (2016) The role of small RNAs in vegetative
helped to collect data. Zeng-Fu Xu conceived the study and shoot development. Curr. Opin. Plant Biol. 29: 64–72.
revised the paper. Fu, Q., Li, C., Tang, M., Tao, Y.-B., Pan, B.-Z., Zhang, L., et al. (2015) An
efficient protocol for Agrobacterium-mediated transformation of the
biofuel plant Jatropha curcas by optimizing kanamycin concentration
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