The PHYB-FOF2-VOZ2 module functions to fine-tune flowering in response to changes in light quality by modulating FLC expression in Arabidopsis.

Proper timing of flowering under different environmental conditions is critical for plant propagation. Light quality is a pivotal environmental cue that plays a critical role in flowering regulation. Plants tend to flower late under light with a high red (R)/far-red (FR) light ratio but early under light with a low R/FR light ratio. However, how plants fine-tune flowering in response to changes in light quality is not well understood. Here, we demonstrate that F-box of Flowering 2 (FOF2), an autonomous pathway-related regulator, physically interacts with VASCULAR PLANT ONE-ZINC FINGER 1 and 2 (VOZ1 and VOZ2), which are direct downstream factors of the R/FR light receptor phytochrome B (PHYB). We show that PHYB physically interacts with FOF2, mediates stabilization of the FOF2 protein under FR light and end-of-day FR light, and enhances FOF2 binding to VOZ2, which leads to degradation of VOZ2 by SCFFOF2 E3 ligase. By contrast, PHYB mediates degradation of FOF2 protein under R light and end-of-day R light. Genetic interaction studies demonstrated that FOF2 functions downstream of PHYB to promote FLC expression and inhibit flowering under both high R/FR light and simulated shade conditions, processes that are partially dependent on VOZ proteins. Taken together, our findings suggest a novel mechanism whereby plants fine-tune flowering time through a PHYB-FOF2-VOZ2 module that modulates FLC expression in response to changes in light quality.

Protocol for detecting lncRNA-protein interaction in vivo using the yeast three-hybrid assay.

Analyses of long non-coding RNA (lncRNA)-protein interactions provide key clues for understanding the molecular basis of lncRNA-modulated biological processes. Here, we detail a yeast three-hybrid assay to identify the lncRNA-interacting protein. We describe steps for lncRNA bait preparation, screening an RNA-binding proteins (RBPs) cDNA library, and validation of the lncRNA-RBP interaction. The assay can also be further applied to delineate the region of RBP that mediates the RNA-protein interaction. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

LncRNA DANA1 promotes drought tolerance and histone deacetylation of drought responsive genes in Arabidopsis.

Although many long noncoding RNAs have been discovered in plants, little is known about their biological function and mode of action. Here we show that the drought-induced long intergenic noncoding RNA DANA1 interacts with the L1p/L10e family member protein DANA1-INTERACTING PROTEIN 1 (DIP1) in the cell nucleus of Arabidopsis, and both DANA1 and DIP1 promote plant drought resistance. DANA1 and DIP1 increase histone deacetylase HDA9 binding to the CYP707A1 and CYP707A2 loci. DIP1 further interacts with PWWP3, a member of the PEAT complex that associates with HDA9 and has histone deacetylase activity. Mutation of DANA1 enhances CYP707A1 and CYP707A2 acetylation and expression resulting in impaired drought tolerance, in agreement with dip1 and pwwp3 mutant phenotypes. Our results demonstrate that DANA1 is a positive regulator of drought response and that DANA1 works jointly with the novel chromatin-related factor DIP1 on epigenetic reprogramming of the plant transcriptome during the response to drought.

Protocol for detecting lncRNA-protein interactions in vitro by tRSA RNA pull-down assay.

Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitro using a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitro binding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Yang et al. (2023).1.

The long non-coding RNA DANA2 positively regulates drought tolerance by recruiting ERF84 to promote JMJ29-mediated histone demethylation.

Tens of thousands of long non-coding RNAs have been uncovered in plants, but few of them have been comprehensively studied for their biological function and molecular mechanism of their mode of action. Here, we show that the Arabidopsis long non-coding RNA DANA2 interacts with an AP2/ERF transcription factor ERF84 in the cell nucleus and then affects the transcription of JMJ29 that encodes a Jumonji C domain-containing histone H3K9 demethylase. Both RNA sequencing (RNA-seq) and genetic analyses demonstrate that DANA2 positively regulates drought stress responses through JMJ29. JMJ29 positively regulates the expression of ERF15 and GOLS2 by modulation of H3K9me2 demethylation. Accordingly, mutation of JMJ29 causes decreased ERF15 and GOLS2 expression, resulting in impaired drought tolerance, in agreement with drought-sensitive phenotypes of dana2 and erf84 mutants. Taken together, these results demonstrate that DANA2 is a positive regulator of drought response and works jointly with the transcriptional activator ERF84 to modulate JMJ29 expression in plant response to drought.

Long noncoding RNA ARTA controls ABA response through MYB7 nuclear trafficking in Arabidopsis.

In eukaryotes, transcription factors are a crucial element in the regulation of gene expression, and nuclear translocation is the key to the function of transcription factors. Here, we show that the long intergenic noncoding RNA ARTA interacts with an importin ß-like protein, SAD2, through a long noncoding RNA-binding region embedded in the carboxyl terminal, and then it blocks the import of the transcription factor MYB7 into the nucleus. Abscisic acid (ABA)-induced ARTA expression can positively regulate ABI5 expression by fine-tuning MYB7 nuclear trafficking. Therefore, the mutation of arta represses ABI5 expression, resulting in desensitization to ABA, thereby reducing Arabidopsis drought tolerance. Our results demonstrate that lncRNA can hijack a nuclear trafficking receptor to modulate the nuclear import of a transcription factor during plant responses to environmental stimuli.

Targeted DNA demethylation of the Arabidopsis genome using the SunTag-dCpf1-TET1cd system.

DNA methylation is a stable and heritable epigenetic mark, and it plays an important role in regulation of gene expression and transposon silencing. Here we developed a CRISPR/dCpf1-based targeted demethylation system using the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and a SunTag system. The SunTag-dCpf1-TET1cd system is able to achieve targeted DNA demethylation and up-regulate gene expression when guided to the FWA or CACTA1 loci in Arabidopsis thaliana . Our study provides tools for targeted removal of DNA cytosine methylation, and activation of protein-coding genes or transposons expression.

Coordinating Diverse Functions of miRNA and lncRNA in Fleshy Fruit.

Non-coding RNAs play vital roles in the diverse biological processes of plants, and they are becoming key topics in horticulture research. In particular, miRNAs and long non-coding RNAs (lncRNAs) are receiving increased attention in fruit crops. Recent studies in horticulture research provide both genetic and molecular evidence that miRNAs and lncRNAs regulate biological function and stress responses during fruit development. Here, we summarize multiple regulatory modules of miRNAs and lncRNAs and their biological roles in fruit sets and stress responses, which would guide the development of molecular breeding techniques on horticultural crops.

Expanding the range of CRISPR/Cas9-directed genome editing in soybean.

The CRISPR/Cas9 system has been widely applied for plant genome editing. The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif (PAM) sequences in the canonical form NGG and non-canonical NAG. Although these PAM sequences are extensively distributed across plant genomes, a broader scope of PAM sequence is required to expand the range of genome editing. Here we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results determined that xCas9 with the NGG and KGA (contains TGA and GGA) PAMs successfully induces genome editing in soybean genome. SpCas9-NG could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. In addition, XNG-Cas9 was observed to cleave soybean genomic regions with NGG, GAA and AGY (contains AGC and AGT) PAM. Moreover, off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency. These findings significantly expand the scope of Cas9-mediated genome editing in soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00051-4.

The long noncoding RNA FRILAIR regulates strawberry fruit ripening by functioning as a noncanonical target mimic.

Long noncoding RNAs (lncRNAs) are emerging as important regulators in plant development, but few of them have been functionally characterized in fruit ripening. Here, we have identified 25,613 lncRNAs from strawberry ripening fruits based on RNA-seq data from poly(A)-depleted libraries and rRNA-depleted libraries, most of which exhibited distinct temporal expression patterns. A novel lncRNA, FRILAIR harbours the miR397 binding site that is highly conserved in diverse strawberry species. FRILAIR overexpression promoted fruit maturation in the Falandi strawberry, which was consistent with the finding from knocking down miR397, which can guide the mRNA cleavage of both FRILAIR and LAC11a (encoding a putative laccase-11-like protein). Moreover, LAC11a mRNA levels were increased in both FRILAIR overexpressing and miR397 knockdown fruits, and accelerated fruit maturation was also found in LAC11a overexpressing fruits. Overall, our study demonstrates that FRILAIR can act as a noncanonical target mimic of miR397 to modulate the expression of LAC11a in the strawberry fruit ripening process.

The Arabidopsis F-box protein FOF2 regulates ABA-mediated seed germination and drought tolerance.

The phytohormone abscisic acid (ABA) plays a crucial role at various plant developmental stages, including seed germination and seedling development, and regulates stomatal aperture in response to drought. However, the underlying mechanisms are not well understood. Here, we showed that F-BOX OF FLOWERING 2 (FOF2) is induced by ABA and drought stress. Overexpression of FOF2 led to reduced ABA sensitivity during seed germination and early seedling development, whereas the fof2 mutant exhibited increased sensitivity to ABA. Molecular and genetic analyses revealed that FOF2 negatively affected ABA-mediated seed germination and early seedling development partially by repressing the expression of the ABA-signaling genes ABI3 and ABI5. Additionally, we found that FOF2-overexpressing plants exhibited increased ABA contents, enhanced ABA sensitivity during stomatal closure, and decreased water loss, thereby improving tolerance to drought stress, in contrast to the fof2 mutant. Consistent with a higher ABA content and enhanced drought tolerance, the expression of ABA- and drought-induced genes and the ABA-biosynthesis gene NCED3 was upregulated in the FOF2-overexpressing plants but downregulated in fof2 mutant in response to drought stress. Taken together, our findings revealed that FOF2 plays an important negative role in ABA-mediated seed germination and early seedling development, as well as a positive role in ABA-mediated drought tolerance.

Mutations in MIR396e and MIR396f increase grain size and modulate shoot architecture in rice.

Grain size and plant architecture are critical factors determining crop productivity. Here, we performed gene editing of the MIR396 gene family in rice and found that MIR396e and MIR396f are two important regulators of grain size and plant architecture. mir396ef mutations can increase grain yield by increasing grain size. In addition, mir396ef mutations resulted in an altered plant architecture, with lengthened leaves but shortened internodes, especially the uppermost internode. Our research suggests that mir396ef mutations promote leaf elongation by increasing the level of a gibberellin (GA) precursor, mevalonic acid, which subsequently promotes GA biosynthesis. However, internode elongation in mir396ef mutants appears to be suppressed via reduced CYP96B4 expression but not via the GA pathway. This research provides candidate gene-editing targets to breed elite rice varieties.

Pod-shattering characteristics differences between two groups of soybeans are associated with specific changes in gene expression.

Soybean is an economically important leguminous crop, and pod dehiscence of soybean could cause huge yield loss. In this study, we measured fruit-cracking forces and percentages of dehisced pods for ten soybean accessions, then separated them into two groups as shattering-sensitive (SS) and shattering-resistant (SR) soybeans. Pod transcriptomes from these two groups were analyzed, and 225 differentially expressed genes (DEGs) were identified between SS and SR soybeans. Some of these DEGs have been previously reported to be associated with pod dehiscence in soybean. The expression patterns of selected DEGs were validated by real-time quantitative reverse transcription PCR, which confirmed the expression changes found in RNA-seq analysis. We also de novo identified 246 soybean pod-long intergenic ncRNAs (lincRNAs), 401 intronic lncRNAs, and 23 antisense lncRNAs from these transcriptomes. Furthermore, genes and lincRNAs co-expression network analysis showed that there are distinct expression patterns between SS and SR soybeans in some co-expression modules. In conclusion, we systematically investigated potential genes and molecular pathways as candidates for differences in soybean pod dehiscence and will provide a useful resource for molecular breeding of soybeans.

A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis.

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.

Ectopic expression of GA 2-oxidase 6 from rapeseed (Brassica napus L.) causes dwarfism, late flowering and enhanced chlorophyll accumulation in Arabidopsis thaliana.

Gibberellins (GAs) are endogenous hormones that play an important role in higher plant growth and development. GA2-oxidase (GA2ox) promotes catabolism and inactivation of bioactive GAs or their precursors. In this study, we identified the GA2-oxidase gene, BnGA2ox6, and found it to be highly expressed in the silique and flower. Overexpression of BnGA2ox6 in Arabidopsis resulted in GA-deficiency symptoms, including inhibited elongation of the hypocotyl and stem, delayed seed germination, and late flowering. BnGA2ox6 overexpression reduced silique growth, but had no effect on seed development. Additionally, BnGA2ox6 overexpression enhanced chlorophyll b and total chlorophyll accumulation, and downregulated mRNA expression levels of the CHL1 and RCCR genes, which are involved in the chlorophyll degradation. These findings suggest that BnGA2ox6 regulates plant hight, silique development, flowering and chlorophyll accumulation in transgenic Arabidopsis.

Construction and Validation of a Dual-Transgene Vector System for Stable Transformation in Plants.

In this study, we constructed dual-transgene vectors (pDT1, pDT7, and pDT7G) that simultaneously co-expressed two genes in plants. ACTIN2 and UBQ10 promoters were used to control the expression of these two genes. The 4×Myc, 3×HA, and 3×Flag reporter genes allowed for the convenient identification of a tunable co-expression system in plants, whereas the dexamethasone (Dex) inducible reporter gene C-terminus of the glucocorticoid receptor (cGR) provided Dex-dependent translocation of the fusion gene between the nucleus and cytoplasm. The function of pDT vectors was validated using four pairwise genes in Nicotiana benthamiana or Arabidopsis thaliana. The co-expression efficiency of two genes from the pDT1 and pDT7G vectors was 35% and 42%, respectively, which ensured the generation of sufficient transgenic materials. These pDT vectors are simple, reliable, efficient, and time-saving tools for the co-expression of two genes through a single transformation event and can be used in the study of protein-protein interactions or multi-component complexes.

The blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.

Arabidopsis cryptochrome 2 (CRY2) is a blue light receptor that mediates light inhibition of hypocotyl elongation and long-day promotion of floral initiation. CRY2 is known to undergo blue light-dependent phosphorylation, which is believed to serve regulatory roles in the function of CRY2. We report here on a biochemical and genetics study of CRY2 phosphorylation. Using mass spectrometry analysis, we identified three serine residues in the CCE domain of CRY2 (S598, S599, and S605) that undergo blue light-dependent phosphorylation in Arabidopsis seedlings. A study of serine-substitution mutations in the CCE domain of CRY2 demonstrates that CRY2 contains two types of phosphorylation in the CCE domain, one in the serine cluster that causes electrophoretic mobility upshift and the other outside the serine cluster that does not seem to cause mobility upshift. We showed that mutations in the serine residues within and outside the serine cluster diminished blue light-dependent CRY2 phosphorylation, degradation, and physiological activities. These results support the hypothesis that blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.