Overexpression of OsHox32 Results in Pleiotropic Effects on Plant Type Architecture and Leaf Development in Rice
- Ying-ying Li†1,
- Ao Shen†1,
- Wei Xiong†1,
- Qiong-lin Sun1,
- Qian Luo1,
- Ting Song1,
- Zheng-long Li1 and
- Wei-jiang Luan1Email author
© The Author(s). 2016
Received: 8 March 2016
Accepted: 6 September 2016
Published: 13 September 2016
The Class III homeodomain Leu zipper (HD-Zip III) gene family plays important roles in plant growth and development. Here, we analyze the function of OsHox32, an HD-Zip III family member, and show that it exhibits pleiotropic effects on regulating plant type architecture and leaf development in rice.
Transgenic lines overexpressing OsHox32 (OsHox32-OV) produce narrow leaves that roll towards the adaxial side. Histological analysis revealed a decreased number of bulliform cells in OsHox32-OV lines. In addition, the angle between the leaf and culm was reduced, resulting in an erect plant phenotype. The height of the plants was reduced, resulting in a semi-dwarf phenotype. In addition, the chlorophyll level was reduced, resulting in a decrease in the photosynthetic rate, but water use efficiency was significantly improved, presumably due to the rolled leaf phenotype. OsHox32 exhibited constitutive expression in different organs, with higher mRNA levels in the stem, leaf sheath, shoot apical meristems and young roots, suggesting a role in plant-type and leaf development. Moreover, OsHox32 mRNA levels were higher in light and lower in the dark under both long-day and short-day conditions, indicating that OsHox32 may be associated with light regulation. Photosynthesis-associated and chlorophyll biosynthesis-associated genes were down-regulated to result in the reduction of photosynthetic capacity in OsHox32-OV lines. mRNA level of six rice YABBY genes is up-regulated or down-regulated by OsHox32, suggesting that OsHox32 may regulate the architecture of plant type and leaf development by controlling the expression of YABBY genes in rice. In addition, OsHox32 mRNA level was induced by the phytohormones, indicating that OsHox32 may be involved in phytohormones regulatory pathways.
OsHox32, an HD-Zip III family member, plays pleiotropic effects on plant type architecture and leaf development in rice.
KeywordsRice (Oryza sativa L.) OsHox32 Overexpression Plant type Rolled leaf
The functions of a leaf, including photosynthesis, respiration and transpiration, are critical for plant survival and are dependent on three-dimensional architecture specific to the plant type (Govaerts et al. 1996). Leaf shape and morphological architecture are considered the most important agronomic traits in rice. Moderate leaf rolling in rice can improve its light capture and gas exchange abilities (Eshed et al. 2001; Moon and Hake. 2011); in addition, appropriate leaf rolling is also related to improved stress responses via reduced direct solar radiation exposure and decreased leaf transpiration under drought stress (Lang et al. 2004; Zhang et al. 2009). Therefore, moderate leaf rolling is highly important for increased grain yield in rice. Recently, several genes regulating the leaf rolling phenotype have been identified and characterized in rice. For example, SHALLOT-LIKE1 (SLL1)/RL9, a transcription factor of the KANADI family, regulates leaf abaxial cell development in rice (Yan et al. 2008; Zhang et al. 2009). sll1 mutants display extremely incurved leaves due to the defective development of sclerenchymatous cells on the abaxial side of the leaf; moreover, the overexpression of SLL1 also resulted in leaf rolling by stimulating phloem development on the abaxial side and suppressing bulliform cell and sclerenchyma development on the adaxial side (Zhang et al. 2009). Adaxialized Leaf 1 (ADL1) encodes a plant-specific calpain-like Cys protease and is required for the establishment of the adaxial-abaxial axis in the leaf primordium (Hibara et al. 2009). adl1 mutants display abaxially rolled leaves due to the increase of bulliform cells on the adaxial side and the formation of bulliform-like cells on the abaxial side of the leaf (Hibara et al. 2009). The overexpression of Abaxially Curled Leaf1 (ACL1) and its homolog ACL2 results in abaxial leaf curling due to increased bulliform cell number and size in rice (Li et al. 2010). Rice outermost cell-specific gene5 (Roc5), a member of homeodomain leucine zipper class IV, also controls leaf rolling by regulating bulliform cell fate and development (Zou et al. 2011). The ectopic expression of Roc5 results in adaxially rolled leaves, whereas cosuppression of Roc5 results in abaxial leaf rolling (Zou et al. 2011). Moreover, several cellulose synthase-like genes and glycosylphosphatidylinositol-anchored proteins have been found to control leaf rolling in rice. The narrow leaf and dwarf1 (ND1)/the curled and dwarf leaf 1 (OsCD1)/the narrow and rolled leaf1 (OsCslD4) encodes a member of the cellulose synthase-like D subfamily. Defects in ND1/OsCD1/OsCslD4 produced dwarfed plants with narrow and rolled leaves due to changes in cell wall composition (Li et al. 2009; Hu et al. 2010; Luan et al. 2011). Furthermore, SEMI-ROLLED LEAF1 (SRL1), a glycosylphosphatidylinositol-anchored protein, was found to regulate the formation of bulliform cells in the adaxial cell layers, leading to leaf rolling in rice (Xiang et al. 2012). Recently, a zinc finger homeodomain class homeobox transcription factor (OsZHD1) was found in rice to induce abaxially curling and drooping due to increased bulliform cell numbers (Xu et al. 2014).
Homeobox genes were originally found in Drosophila to control the development of body segments (Scott et al. 1989). Thus far, they have been identified in different organisms, including various animal species, yeast, fungi, and higher plants. Homeobox genes contain a conserved DNA-binding motif known as the homeodomain that consists of 60 amino acid residues. In higher plants, a homeodomain superfamily with a closely linked leucine zipper motif, named HD-Zip, was first discovered in Arabidopsis thaliana (Ruberti et al. 1991). At present, HD-Zips have been identified in plants such as sunflower (Chan and Gonzalez 1994), soybean (Moon et al. 1996), carrot (Kawahara et al. 1995), tomato (Meissner and Theres 1995; Tornero et al. 1996), and rice (Meijer et al. 1997; Itoh et al. 2008; Luan et al. 2013). Based on differences of gene structure, motifs, and specific DNA binding sequence (Sessa et al. 1998), HD-Zip members can be divided into four groups, HD-Zip I through HD-Zip IV. All HD-Zip proteins function as mediators of plant development. Five members of the HD-Zip III, PHABULOSA (PHB), PHAVOLUTA (PHV), REVOLUTA (REV), CORONA (CNA), and AtHB8 in Arabidopsis, serve partially redundant and antagonistic roles in the development of Arabidopsis (Emery et al. 2003; Prigge et al. 2005). PHB, PHV, REV and CNA are related to the initiation of the shoot apical meristem (SAM) as well as the formation of axillary SAMs (Talbert et al. 1995; Otsuga et al. 2001; Emery et al. 2003; Green et al. 2005; Prigge et al. 2005), whereas AtHB8 is implicated in vascularization (Baima et al. 2001). In addition, REV, PHB and PHV can interact with KANADI to regulate the abaxial-adaxial patterning of lateral organs via feedback mechanisms. KANADI is required for the formation of abaxial tissues, but its expression represses that of REV, PHB and PHV (Emery et al. 2003). Recently, studies have shown that HD-Zip III members are regulated by microRNAs such as miRNA165 and miRNA166 (Kim et al. 2005; Williams et al. 2005; Mallory and Vaucheret 2006). Mutation of these two microRNAs gives rise to HD-Zip III gain-of-function phenotypes in Arabidopsis. In rice, the expression patterns and ectopic expression phenotypes of five HD-Zip III members, OsHB1-OsHB5, were analyzed, suggesting that they were essential for radial pattern formation during embryogenesis and the leaf initiation process in the shoot apical meristem (Itoh et al. 2008). However, they only analyzed the effect of ectopic expression of OsHB1, OsHB3, and OsHB5 in Itoh et al.’ study, but not OsHB4 (OsHox32). In this study, we analyzed the pleiotropic functions of OsHox32 in the development and architecture of rice plant type and leaf. Using reverse genetics, we demonstrate that overexpression of OsHox32 leads to leaf rolling and semi-dwarf phenotype in rice.
OsHox32 is a Member of the HD-Zip III Family
Overexpression of OsHox32 Affects the Architecture of the Rice Leaf and Plant Type
Overexpression of OsHox32 Affects Photosynthesis Indexes and Water use Efficiency
The Expression Pattern of OsHox32
Subcellular Localization of OsHox32
Effect of Phytohormone Signal on OsHox32 Expression
Photosynthesis-Associated and Chlorophyll Biosynthesis-Associated Genes Were Down-Regulated in OsHox32-OV Plants
OsHox32 Regulates the Expression of YABBY Genes to Control the Architecture of Plant Type and Leaf in Rice
In addition, we analyzed the expression of two rolled leaf-related genes in OsHox32-OV plants and found that the expression of Roc5 (encoding a member of homeodomain leucine zipper Class IV) and SRL1 (encoding a glycosylphosphatidylinositol-anchored protein) did not significantly differ between OsHox32-OV and wild type plants (Fig. 8g, h), indicating that OsHox32 does not regulate the expression of Roc5 and SRL1.
Pleiotropic Effects of OsHox32 on the Regulation of Plant Growth and Development
Members of the Class III homeodomain-Leu zipper family play an important role in determining plant morphological architecture. In Arabidopsis, there are five Class III HD-Zip genes: REV, PHB, PHV, CNA, and ATHB8. Phenotypic analysis of loss-of-function mutants of these genes showed that they play distinct overlapping and antagonistic roles in development. PHB, PHV, and REV play overlapping and crucial roles during the establishment of apical bilateral symmetry and leaf abaxial/adaxial polarity (Prigge et al. 2005); rev phv double mutants displayed a trumpet-shaped leaf. REV plays an important role in the formation of the floral meristem. rev mutants display defective floral organs and produce sterile flowers. CNA and ATHB8 exhibit antagonistic functions toward REV during the formation of the lateral shoot meristem and the floral meristem, which can restore the partial phenotype of rev mutants (Prigge et al. 2005). In maize, RLD1 encodes an HD-ZipIII protein whose adaxial expression is defined by miRNA166-directed transcript cleavage on the abaxial side, which is an important determinant of adaxial cell fate during maize leaf development (Juarez et al. 2004). In Populus trichocarpa, eight HD-Zip III genes have been identified (Ko et al. 2006; Côté et al. 2010). PRE, a homolog of the Arabidopsis REV gene, is related to the initiation of the cambium and the pattern of secondary vascular tissues (Robischon et al. 2011). Another member, PCN, may negatively regulate secondary vascular cell differentiation (Kim et al. 2005; Du et al. 2011). In addition, PtrHB7, an ortholog of AtHB8 in Arabidopsis, plays an essential role in the regulation of balanced differentiation between secondary xylem and phloem tissues during the process of secondary growth in Populus. These previous studies provide evidence that HD-Zip III members have evolved distinct functions and exert different pleiotropic effects on growth and development in different plants.
In rice, there are five HD-Zip class III members: OsHox9, OsHox10, OsHox29, OsHox32, and OsHox33, corresponding to OsHB1-OsHB5 as described by Itoh et al. (2008). In a study by Itoh et al., the effect of ectopic expression of OsHB1, OsHB3, and OsHB5, but not OsHB4 (OsHox32) was analyzed; overexpression of these genes resulted in abnormalities such as rolled and filamentous leaves. In this study, we investigated the role of OsHox32, which corresponds to OsHB4. Our results showed that OsHox32 regulates the morphological architecture of the rice leaf. Overexpression of OsHox32 resulted in narrow and upward rolling leaves. Further histological observation showed that the number of bulliform cells decreased in OsHox32-OV lines. This evidence suggests that OsHox32 has a conserved function in the regulation of the morphological architecture and development of the rice leaf. However, OsHox32 also regulates the architecture of plant type and the photosynthetic capacity in rice except the regulation of the morphological architecture of the rice leaf. Our results demonstrated that overexpression of OsHox32 resulted in semi-dwarf phenotype. Expressed analysis found that mRNA level of six rice YABBY genes is up-regulated or down-regulated by OsHox32. This result is in agreement with exhibiting semi-dwarf phenotype of OsYABBY1 overexpressing. In addition, our previous study revealed that OsHox33 plays an important role in leaf senescence (Luan et al. 2013). Therefore, HD-Zip III members have evolved distinct functions except a conserved function in the regulation of the leaf architecture in rice.
OsHox32 may Regulate the Leaf and Plant Type Architecture by Controlling YABBY Genes in Rice
In Arabidopsis, the YABBY genes play important roles in establishment of adaxial-baxial polarity, floral organ development and lamina formation. Loss of function of YABBY genes results in loss of polar differentiation of abaxial cells in the lateral organs and the defect of floral organ (Sawa et al. 1999; Sarojam et al. 2010). In rice, identified YABBY members act as crucial function in leaf, the floral organ and vascular development and plant type architecture. Overexpression of OsYABB1 caused semi-dwarf phenotype, whereas cosuppression of OsYABB1 induced rolled leaf phenotype (Dai et al. 2007b). OsYABBY3 was expressed in the leaf and floral organ primordial, playing important roles in leaf cell growth and differentiation (Dai et al. 2007a). Knockdown of OsYABBY3 resulted in twisted and knotted leaves. OsYABBY4 was expressed in the meristems and developing vascular tissue of rice, without an adaxial/abaxial polar expression pattern in lateral organs, suggesting that OsYABBY4 might play an important role in vascular development (Liu et al. 2007). OsYABBY5/TOB1 was expressed in the lateral organs primordia, but not in the meristem, playing important roles for the initiation and growth of the lemma and palea (Tanaka et al. 2012). Knockdown of OsYABBY5 resulted in the defect of spikelet, overexpression of OsYABBY5 induced an excess number of floral organs such as pistils, stamens, and lodicules (Tanaka et al. 2012). These previous reports showed that the up- or down-regulation of OsYABBY genes seriously affected leaf development and plant type architecture. Our result indicated that OsYABBY genes were up- or down-regulated in OsHox32 overexpressing plants. Moreover, the overexpression of OsHox32 resulted in rolled leaf and semi-dwarf phenotypes. Taken together the previous studies and our results, we proposed that OsHox32 might regulate the architecture of plant type and leaf development by controlling the expression of YABBY genes in rice.
The Subcellular Localization of OsHox32
Members of HD-Zip III family contain a DNA-binding motif and exhibit transcription factor activity (Ariel et al. 2007). Many transcription factors, such as GmERF5 in soybean, TaWRKY10 in wheat, and OsMYB103L in rice, were localized in the nucleus (Hernandez-Garcia et al. 2010; Wang et al. 2013; Yang et al. 2014). In addition, the transcription factor Roc5, which plays a role in regulating the formation of rice leaves, was also localized in the nucleus (Zou et al. 2011). However, not all transcription factors are localized in nucleus. The heat-shock transcription factor AtHsfA6a, which abnormally acts as a transcriptional activator of stress-responsive genes via the ABA-dependent signaling pathway, was found in the cytoplasm and nucleus simultaneously (Hwang et al. 2014). Moreover, the transcription factor LeERF1 in tomato was also localized both in the cytoplasm and nucleus (Zhang et al. 2012). In our study, the results of transient expression showed that OsHox32 was localized in the cell periphery, but no significant signal was detected in the nucleus. We speculate that OsHox32 may be a cytoplasmic protein because the cytoplasm in mature plant epidermal cells is restricted to the cell periphery due to the presence of a large central vacuole. This result is supported by the prediction by PSOT software and the Rice Genome Annotation Database (http://ricegaas.dna.affrc.go.jp/).
Effect of Phytohormone Treatments on HD-Zip III Family Gene Expression
In Arabidopsis, HD-Zip III gene expression patterns are correlated with auxin distribution patterns in shoot meristem and in meristematic tissues of the vasculature (McConnell et al. 2001; Otsuga et al. 2001; Emery et al. 2003; Prigge et al. 2005). Moreover, HD-Zip III proteins can promote axial cell elongation and xylem differentiation via auxin pathways (Ilegems et al. 2010). The recently reported DRN-like (DRNL) protein interacts with HD-Zip III members to control the balance of cell proliferation and differentiation in meristematic tissues via interactions with auxin signaling (Chandler et al. 2007). In rice, members of HD-Zip III family are highly expressed in the SAM flank at an early stage of leaf initiation, then disappear from the flank in the middle stage of leaf primordium development (Itoh et al. 2008). This change is similar to that of the leaf determination process, which is regulated by local auxin accumulation in the SAM. To reveal whether OsHox32 responds to phytohormone pathways, we treated wild-type plants with IAA, JA, ACC or ABA and monitored OsHox32 expression by real-time PCR. We found that the expression of OsHox32 was induced by phytohormone treatment, indicating that OsHox32 may be involved in phytohormones regulatory pathways. However, the pattern of induction varied between IAA and JA, ACC, ABA. Under normal conditions (ie, no treatments were performed), OsHox32 exhibits higher expression during the light stage and lower expression during the dark stage due to the influence of the circadian clock. However, the pattern of OsHox32 expression is changed after IAA treatment; the expression of OsHox32 remained higher during the dark stage (12 h after treatment). In contrast, the pattern of OsHox32 expression after treatment with JA, ACC, or ABA is consistent with the pattern of expression under normal conditions. These results indicate that JA, ACC and ABA have different effects on the expression of OsHox32 compared with auxin.
The Function of OsHox32 in Photosynthesis
It has been reported that leaf photosynthesis is affected in several mutants with a rolled leaf phenotype. SLL1 encodes a SHAQKYF class MYB family transcription factor (Zhang et al. 2009). sll1 mutants display extremely rolled leaves of a deeper shade of green. The chlorophyll content and Fv/Fm responding to the photosynthetic capacity were elevated in sll1-1 and sll1-2 plants due to increased numbers of mesophyll cells, suggesting increased photosynthesis due to SLL1 deficiency (Zhang et al. 2009). The photosynthetic efficiency was also improved in the roc5(out1) mutant due to an increase in the stomatal conductance and photosynthetic rate (Zou et al. 2011). Our result showed that the chlorophyll content was significantly reduced, resulting in the reduction of photosynthetic rate in OsHox32-OV plants. qRT-PCR experiments suggested that the expression of photosynthesis-associated and chlorophyll biosynthesis-associated genes was down-regulated in the OsHox32-OV line, indicating that the overexpression of OsHox32 affects photosynthetic capacity. In addition, OsHox32 is more highly expressed during the light stage, suggesting that OsHox32 may be associated with the regulation of light. Taken together, we speculate that OsHox32 may play an important role in photosynthesis.
In this study, we analyze the function of OsHox32. Overexpression of OsHox32 leads to leaf rolling and semi-dwarf phenotype in rice, indicating that OsHox32 plays pleiotropic effects on plant type architecture and leaf development in rice. OsHox32 exhibited constitutive expression in different organs, with higher mRNA levels in the stem, leaf sheath, shoot apical meristems and young roots, suggesting a role in plant-type and leaf development. In addition, OsHox32 mRNA levels were higher in light and lower in the dark, indicating that OsHox32 may be associated with light regulation. Photosynthesis-associated and chlorophyll biosynthesis-associated genes were down-regulated to result in the reduction of photosynthetic capacity in OsHox32-OV lines. The expression of YABBY genes is up-regulated or down-regulated by OsHox32, suggesting that OsHox32 may regulate the architecture of plant type and leaf development by controlling the expression of YABBY genes in rice.
The rice variety Nipponbare (Oryza sativa L.ssp. japonica) and transgenic plants overexpressing OsHox32 were used in this study. The transgenic plants were generated from Nipponbare using the Agrobacterium-mediated transformation method described by Hiei et al. (1994). Plants were grown in the experimental field at the Tianjin Agricultural Academy of Science, Tianjin, and in Lingshui, Hainan Province, China.
Construction of the Overexpression Vector and Rice Transformation
OsHox32 cDNA was amplified from 15-day-old seedlings by RT-PCR using specific primers: OHox32F: 5’ - TTAAggtaccGAGAAGAAGGAGAAGGGTCG-3’ and OHox32R: 5’ - GAGCtctagaCTCAGACGAATGACCAGTTG-3’. The italic bases are recognition sites for restriction endonuclease enzymes. The resulting fragment was inserted into an empty pCAMBIA2300 binary vector with double CaMV 35S promoters to obtain the resultant vector. After confirmation by sequencing, the resultant vector was introduced into wild-type plants using the Agrobacterium-mediated transformation procedure described by Hiei et al. (1994).
RT-PCR and Real-Time PCR Analysis
For analysis of gene expression in transgenic lines, RNA was isolated from 50-day-old leaves using Trizol reagent (Invitrogen, USA) and treated with DNase I (NEB, USA). For expression analysis of OsHox32 in different organs, RNA was isolated from shoot apical meristems (SAM), young roots, mature leaf blades, leaf sheathes, stems of the first internode, and young panicles at different periods. For expression analysis of different development stages, penultimate leaves were collected at the desired stages for analysis of the expression of OsHox32. cDNA was synthesized from 2 μg total RNA using M-MLV reverse transcriptase (TaKaRa, Dalian, China). One microliter of cDNA was used for RT-PCR analysis with gene-specific primers. OsHox32 amplification conditions were as follows: 2 min at 95 °C; followed by 32 cycles of 30 s at 94 °C, 30 s at 57 °C, 30 s at 72 °C; and then a final extension for 5 min at 72 °C. For OsActin1: 2 min at 95 °C; followed by 24 cycles of 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C; and then a final step of 5 min at 72 °C. Three replicates were carried out for each reaction. Real-time PCR was performed using 1 μl cDNA and SYBR Green PCR master mix (Tiangen, Beijing, China) in a MyiQ2 two-color real-time PCR detection system (Bio-Rad, USA). The 2-△△Ct method described by Livak (Livak and Schmittgen 2001) was used for the analysis of relative gene expression. Three replicates were performed for each reaction, and OsActin1 was used as an internal control for the relative quantification of target gene expression. All primer sequences are provided in Additional file 1: Table S1. The amplification conditions were as follows: 2 min at 95 °C; followed by 40 cycles of 20 s at 95 °C, 30 s at 60 °C, and 30 s at 68 °C.
Expression Analysis of OsHox32 During Different Photoperiods
Strong wild type seedlings approximately 3 weeks old were transferred from natural fields to artificial climate cabinets (MMM, Climacell, Germany). Two photoperiods were used in the artificial climate cabinets with the following parameters: Long-day condition (LD), 15 h light and 9 h dark at 28 °C; short-day condition (SD), 9 h light and 15 h dark at 28 °C. After 22 days in artificial climate cabinets, RNA was isolated from the leaves once every 3 h for 24 h using Trizol solution (Invitrogen, USA). qRT-PCR was used to analyze the expression of OsHox32 using the same method described as above.
Histological Analysis and Leaf Rolling Index
Approximately 60-day-old leaves were collected from wild-type and transgenic plants and were fixed in an FAA solution (70 % ethanol, 5 % formaldehyde and 5 % acetic acid) for 48 h. After dehydration via ethanol series, the samples were infiltrated and embedded in paraffin. 10 μm thick sections were cut using a rotary microtome (Leica RM2235; Leica microsystems, Nussioch, Germany), stained with toluidine blue, and observed under a light microscope (eclipse 80i; Nikon, Tokyo, Japan). The width of leaves in the late booting stage was measured to calculate the leaf rolling index (LRI). The widest position at the middle of every leaf was measured. We measured the leaf width under natural rolling conditions (Ln) and while unfolding from rolling (Lw), and the LRI was calculated as LRI(%) = (Lw-Ln)/Lw × 100 %. The leaf area index was measured using a leaf area meter (CI-203 Area Meter, CID, Inc. USA).
Measurement of Photosynthesis Indexes and Chlorophyll Content
In the field on a sunny morning, the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), and water use efficiency (WUE) were measured in the penultimate and antepenultimate leaves of rice plants during the booting stage using a portable photosynthesis system equipped with an LED (light-emitting diode) light source 6400–02 (LiCor-6400; LiCor Inc. Lincoln, Nebraska, USA). Chlorophyll was extracted from 0.2 g fresh leaves at the booting stage using 80 % acetone. Briefly, leaves were cut into 3 mm pieces and immersed 80 % acetone for 48 h in the dark at 26 °C. The absorbance of the extract was measured using a spectrophotometer at A645 and A663. The chlorophyll a, chlorophyll b and total chlorophyll contents were determined by the method reported by Arnon (1949). A total of 10 individuals of the T2 transgenic line (OsHox32-OV2) and wild type plants, respectively, were assayed.
The Subcellular Localization of OsHox32
The full region encoding of OsHox32 without a stop codon was amplified from 30-day-old seedling cDNA by RT-PCR using gene-specific primers: G-Hox32F: 5’-ATAgagctcGAGAAGAAGGAGAAGGGTCG-3’ and G-Hox32R: 5’- CTGtctagaGACGAATGACCAGTTGACGA-3’. The italic bases are restriction endonuclease recognition sites. The amplified cDNA fragment was inserted into a pCAMBIA35S-GFP empty vector and fused in-frame with green fluorescent protein (GFP) to produce the resultant vector p35S::OsHox32-GFP. After confirmation by sequencing, the fused vector and empty vector were transiently transformed. To analyze tobacco epidermal cells, the fusion vector and empty vector were introduced into the Agrobacterium tumefaciens strain and injected into tobacco (Nicotiana tabacum) leaves by infiltration of the Agrobacterium tumefaciens strain. After culturing for 2–3 days, the tobacco epidermis was visualized using a laser confocal microscope (Nikon EZ-C1 Si laser confocal microscope, Japan). For onion transient expression analysis, the fusion vector p35S::OsHox32-GFP and empty vector were transformed into onion (Allium cepa) epidermal cells by gene bombardment (the Bio-Rad PDS-1000/He device, USA). Bombarded epidermal cells were incubated for 2–3 days at 25 °C in the dark. The cell layers were then examined using a laser confocal microscope (Nikon EZ-C1 Si laser confocal microscope, Japan).
Nipponbare (Oryza sativa L. ssp. Japonica) was planted in a greenhouse (28 °C, 14 h light, 10 h dark), and 4-week-old seedlings were subjected to different treatments. Phytohormone treatments were performed by spraying with IAA (0.1 mM), JA (0.1 mM), ACC (0.1 mM) and ABA (0.1 mM) solutions, and water was sprayed as a control. Leaves were collected for total RNA isolation to analyze OsHox32 expression at 0, 1, 3, 6, 12 and 24 h, respectively.
We thank Dr. Sheila McCormick (Plant Gene Expression Center, USDA/ARS and UC-Berkeley) for editing and advice. We also thank Dr. Lina Pan (Tianjin Normal University) for the help of figures editing. This research was supported by the National Natural Science Foundation of China (No. 31171515), the Tianjin Natural Science Foundation of China (No. 16JCZDJC33400 and No. 11JCZDJC17900), and the Knowledge Innovation and Training Program of Tianjin (Tianjin Municipal Education Commission; No. ZX110GG017).
LY, AS and WL designed the research project. LY, AS, WX, QS and WL designed and performed all of the experiments, analyzed the data, and drafted the manuscript. QL, TS, ZL and assisted the transgenic plants, the field experiments and the analysis of the data. All authors read and approved the final manuscript.
YL, AS, QS, TS, ZL and WX are the postgraduates of College of Life Sciences, Tianjin Normal University; QL is the undergraduate of Tianjin Normal University; WL is the professor of College of Life Sciences, Tianjin Key Laboratory of Animal and Plant Resistance (TKL), Tianjin Normal University; responsible for overall work of Lab. Teaching genetic and molecular biology.
All authors declare that they have no competing interests.
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