Genetic Transformation of LoHDZ2 and Analysis of its Function to Enhance Stress Resistance in Larch

To study the function of LoHDZ2 in larch, we first constructed a p1302-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG6000 and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured.The results are summarized below.1. When the specific fragment of the target gene in the genome of the resistant cell line was amplified, at the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines.2. To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 downregulated ones) were subjected to qRT–PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-seq, indicating that the transcriptome data were reliable.3. The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were downregulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process.4. After PEG6000 treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced.In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2, which is a member of the HD-ZipII subfamily, of Larix olgensis may participate in the response of plants to the external environment and may participate in the growth and development of Larix olgensis by affecting plant metabolic pathways.

GhD14 Regulates Plant Architecture and Fiber Development in Cotton

Background: Strigolactone (SL) signaling is essential in regulating plant development. DWARF14 (D14), the SL receptor, interacts with the F-box in MORE AXILLARY GROWTH (MAX2) to modulate SL signaling. However, the biological function of D14 protein is still unknown in cotton.Results: Here, we identified GhD14s in Gossypium hirsutum and resolved its function in cotton plant architecture and fiber development. Subcellular location results revealed that the GhD14D protein was localized to both the cytoplasm and nucleus. GUS staining assay showed that GhD14D was mainly expressed in leaf primordium, inflorescence, axillary bud and stem and expression analysis revealed that GhD14A/D was highly expressed in stem, flower and fiber cells at 20 days post-anthesis (DPA). Silencing GhD14A/D gene expression in upland cotton significantly increased branch angle. Meanwhile, the fiber length and the transcripts of secondary cell wall biosynthesis related genes were also reduced after GhD14A/D gene silencing. In addition, overexpression of GhD14D in Atd14 mutant successfully rescued the phenotype of the d14 mutant with much shoot-branching and short plant height.Conclusions: Our findings suggest that the GhD14 gene contributes to shoot branch development and fiber cell development in cotton. This study deepens our understanding of the biological role of SL signaling in cotton and providing guidance for modifying cotton plant architecture and improving fiber development using genetic engineering to help us breed better cotton varieties in the future.

GUS staining protocol v1

All the reagents for GUS staining are easy to use. Just mix the prepared X-gluc solution and buffer in proportion to form a GUS staining solution. This kit can prepare 50ml GUS staining solution.

Isolation and Characterization of APETALA3 Orthologs and Promoters from the Distylous Fagopyrum esculentum

Common buckwheat (Fagopyrum esculentum) produces distylous flowers with undifferentiated petaloid tepals, which makes it obviously different from flowers of model species. In model species Arabidopsis, APETALA3 (AP3) is expressed in petal and stamen and specifies petal and stamen identities during flower development. Combining with our previous studies, we found that small-scale gene duplication (GD) event and alternative splicing (AS) of common buckwheat AP3 orthologs resulted in FaesAP3_1, FaesAP3_2 and FaesAP3_2a. FaesAP3_2 and FaesAP3_2a were mainly expressed in the stamen of thrum and pin flower. Promoters functional analysis suggested that intense GUS staining was observed in the whole stamen in pFaesAP3_2::GUS transgenic Arabidopsis, while intense GUS staining was observed only in the filament of stamen in pFaesAP3_1::GUS transgenic Arabidopsis. These suggested that FaesAP3_1 and FaesAP3_2 had overlapping functions in specifying stamen filament identity and work together to determine normal stamen development. Additionally, FaesAP3_2 and FaesAP3_2a owned the similar ability to rescue stamen development of Arabidopsis ap3-3 mutant, although AS resulted in a frameshift mutation and consequent omission of the complete PI-derived motif and euAP3 motif of FaesAP3_2a. These suggested that the MIK region of AP3-like proteins was crucial for determining stamen identity, while the function of AP3-like proteins in specifying petal identity was gradually obtained after AP3 Orthologs acquiring a novel C-terminal euAP3 motif during the evolution of core eudicots. Our results also provide a clue to understanding the early evolution of the functional specificity of euAP3-type proteins involving in floral organ development in core eudicots, and also suggested that FaesAP3_2 holds the potential application for biotechnical engineering to develop a sterile male line of F. esculentum.

The transcription factor AtGLK1 acts upstream of MYBL2 to genetically regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis

Background The regulation of anthocyanin biosynthesis by various factors including sugars, light and abiotic stresses is mediated by numerous regulatory factors acting at the transcriptional level. Here experimental evidence was provided in order to demonstrate that the nuclear GARP transcription factor AtGLK1 plays an important role in regulating sucrose-induced anthocyanin biosynthesis in Arabidopsis. Results The results obtained using real-time quantitative PCR and GUS staining assays revealed that AtGLK1 was mainly expressed in the green tissues of Arabidopsis seedlings and could be induced by sucrose. The loss-of-function glk1 glk2 double mutant has lower anthocyanin levels than the glk2 single mutant, although it has been determined that loss of AtGLK1 alone does not affect anthocyanin accumulation. Overexpression of AtGLK1 enhances the accumulation of anthocyanin in transgenic Arabidopsis seedlings accompanied by increased expression of anthocyanin biosynthetic and regulatory genes. Moreover, we found that AtGLK1 also participates in plastid-signaling mediated anthocyanin accumulations. Genetic, physiological, and molecular biological approaches demonstrated that AtGLK1 acts upstream of MYBL2, which is a key negative regulator of anthocyanin biosynthesis, to genetically regulate sucrose-induced anthocyanin biosynthesis. Conclusion Our results indicated that AtGLK1 positively regulates sucrose-induced anthocyanin biosynthesis in Arabidopsis via MYBL2.

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying GUS genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

GhD14 regulates plant architecture and fiber development in cotton

Strigolactone (SL) signaling is essential in regulating plant development. DWARF14 (D14), the SL receptor, interacts with the F-box in MORE AXILLARY GROWTH (MAX2) to modulate SL signaling. However, the biological function of D14 protein is still unknown in cotton. Here, we identified GhD14s in Gossypium hirsutum and resolved its function in cotton plant architecture and fiber development. The GhD14D protein was localized to both the cytoplasm and nucleus. GUS staining assay showed that GhD14D was mainly expressed in leaf primordium, inflorescence, axillary bud and stem and expression analysis revealed that GhD14A/D was highly expressed in stem, flower and fiber cells at 20 days post-anthesis (DPA). Silencing GhD14A/D gene expression in upland cotton significantly increased branch angle and reduced fiber length as well as the transcripts of secondary cell wall biosynthesis related genes. In addition, overexpression of GhD14D in Atd14 mutant successfully rescued the phenotype of the d14-1 mutant with much shoot-branching and short plant height. Our findings suggest that the GhD14 gene contributes to shoot branch development and fiber cell development in cotton. This study deepens our understanding of the biological role of SL signaling in cotton and providing guidance for modifying cotton plant architecture and improving fiber development using genetic engineering to help us breed better cotton varieties in the future.

The Transcription Factor AtGLK1 Acts Upstream of MYBL2 to Genetically Regulate Sucrose-Induced Anthocyanin Biosynthesis in Arabidopsis

Background: The regulation of anthocyanin biosynthesis by various factors including sugars, light and abiotic stresses is mediated by numerous regulatory factors acting at the transcriptional level. Here experimental evidence was provided in order to demonstrate that the nuclear GARP transcription factor AtGLK1 plays an important role in regulating sucrose-induced anthocyanin biosynthesis in Arabidopsis.Results: The results obtained using real-time quantitative PCR and GUS staining assays revealed that AtGLK1 was mainly expressed in the green tissues of Arabidopsis seedlings and could be induced by sucrose. The loss-of-function glk1 glk2 double mutant has lower anthocyanin levels than the glk2 single mutant, although it has been determined that loss of AtGLK1 alone does not affect anthocyanin accumulation. Overexpression of AtGLK1 enhances the accumulation of anthocyanin in transgenic Arabidopsis seedlings accompanied by increased expression of anthocyanin biosynthetic and regulatory genes. Moreover, we found that AtGLK1 also participates in plastid-signaling mediated anthocyanin accumulations. Genetic, physiological, and molecular biological approaches demonstrated that AtGLK1 acts upstream of MYBL2, which is a key negative regulator of anthocyanin biosynthesis, to genetically regulate sucrose-induced anthocyanin biosynthesis.Conclusion: Our results indicated that AtGLK1 positively regulates sucrose-induced anthocyanin biosynthesis in Arabidopsis via MYBL2.

RNAi silencing of the Arabidopsis thaliana ULCS1 gene results in pleiotropic phenotypes during plant growth and development

WD40-repeat-containing proteins (WDRs) are highly abundant in all eukaryotes. Several of them have been implicated as subunits of multi-protein CRL E3 ligase complexes that regulate ubiquitination mediated protein degradation and thus various cellular and developmental processes. Impairment of the WDR protein ULCS1 from Arabidopsis causes pleiotropic phenotypes during plant development, including reduced lignification, anther indehiscence, and sterility. Here we show that RNAi-mediated downregulation of ULCS1 results in a fast-growing phenotype during vegetative development. Due to accelerated growth, ulcs1i mutants reach their vegetative to reproductive transition point earlier than WT plants. However, their comparable germination rate and their similar number of secondary branches and rosette leaves at bolting indicate that ulcs1i is not an early flowering time mutant. GUS staining of progeny, obtained from crosses between ulcs1i and CYCB1::GUS plants, revealed an increased number of mitotic cell divisions in the root meristems of ulcs1i compared to WT. Immunolabeling of homogalacturonans (HGAs) epitopes showed significant fluorescent signal differences at the cell walls and the mucilage of the seeds between ulcs1i and WT. Furthermore, we demonstrate that ULCS1 interacts with the UBA-like protein in a yeast two-hybrid assay, suggesting a direct or indirect physical coupling of these proteins in Arabidopsis.

RGG1, Involved in the Cytokinin Regulatory Pathway, Controls Grain Size in Rice

Heterotrimeric GTP binding proteins (G proteins) and cytokinin play important roles in regulating plant growth and development. However, little is known about the mechanism by which they coordinate the regulation of grain size in rice. We functionally characterized one gene, RGG1, encoding a type-A Gγ subunit. Strong GUS staining was detected in young panicles and spikelets, suggesting a role for this gene in modulating panicle-related trait development. Overexpression of RGG1 in Nipponbare (NIP) and Wuyunjing 30 (WYJ30) significantly decreased plant height, panicle length and grain length by regulating cell division. However, rgg1 mutants generated by the CRISPR/Cas9 system exhibited no obvious phenotypic differences, which may be due to the extremely low expression level of this gene in vivo. The transcriptomes of young panicles of NIP, the NIP-rgg1–2 mutant and the NIP-OE2 overexpression line were sequenced, and the results showed that many differentially expressed genes (DEGs) were associated with the cytokinin biosynthetic pathway. We confirmed this result by measuring the endogenous cytokinin levels and found that cytokinin content was lower in the overexpression lines. Additionally, increased expression of RGG1 decreased sensitivity to low concentrations of 6-benzylaminopurine (6-BA). Our results reveal a novel G protein—cytokinin module controlling grain size in rice and will be beneficial for understanding the mechanisms by which G proteins regulate grain size and plant development.