S-RNase is the female determinant of self-incompatibility (SI) in pear (Pyrus bretschneideri). After translocation to the pollen tube, S-RNase degrades rRNA and induces pollen tube death in an S-haplotype-specific manner. In this study, we found that the actin cytoskeleton is a target of P. bretschneideri S-RNase (PbrS-RNase) and uncovered a mechanism that involves phosphatidic acid (PA) and protects the pollen tube from PbrS-RNase cytotoxicity. PbrS-RNase interacts directly with PbrActin1 in an S-haplotype-independent manner, causing the actin cytoskeleton to depolymerize and promoting programmed cell death in the self-incompatible pollen tube. Proline 156 of PbrS-RNase is essential for the PbrS-RNase-PbrActin1 interaction, and the actin cytoskeleton-depolymerizing function of PbrS-RNase does not require its RNase activity. PbrS-RNase cytotoxicity enhances the expression of phospholipase D (PbrPLDδ1), resulting in increased PA levels in the incompatible pollen tube. PbrPLDδ1-derived PA initially prevents depolymerization of the actin cytoskeleton elicited by PbrS-RNase and delays the SI signaling that leads to pollen tube death. This work provides insights into the orchestration of the S-RNase-based SI response, in which increased PA levels initially play a protective role in incompatible pollen, until sustained PbrS-RNase activity reaches the point of no return and pollen tube growth ceases.

Flowers open at sunrise and close at sunset, establishing a circadian floral movement rhythm to facilitate pollination as part of reproduction. By the coordination of endogenous factors and environmental stimuli, such as circadian clock, photoperiod, light and temperature, an appropriate floral movement rhythm has been established; however, the underlying mechanisms remain unclear. In our study, we use waterlily as a model which represents an early-diverging grade of flowering plants, and we aim to reveal the general mechanism of flower actions. We found that the intermediate segment of petal cells of waterlily are highly flexible, followed by a circadian cell expansion upon photoperiod stimuli. Auxin causes constitutively flower opening while auxin inhibitor suppresses opening event. Subsequent transcriptome profiles generated from waterlily’s intermediate segment of petals at different day-time points showed that auxin is a crucial phytohormone required for floral movement rhythm via the coordination of YUCCA-controlled auxin synthesis, GH3-mediated auxin homeostasis, PIN and ABCB-dependent auxin efflux as well as TIR/AFB-AUX/IAA- and SAUR-triggered auxin signaling. Genes involved in cell wall organization were downstream of auxin events, resulting in the output phenotypes of rapid cell expansion during flower opening and cell shrinkage at flower closure stage. Collectively, our data demonstrate a central regulatory role of auxin in floral movement rhythm and provide a global understanding of flower action in waterlily, which could be a conserved feature of angiosperms.

Background: MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression by targeting messenger RNAs (mRNAs) and causing mRNA cleavage or translation blockage. Of the 355 Arthropod miRNAs that have been identified, only 21 are B. mori miRNAs that were predicted computationally; of these, only let-7 has been confirmed by Northern blotting. Results: Combining a computational method based on sequence homology searches with experimental identification based on microarray assays and Northern blotting, we identified 46 miRNAs, an additional 21 plausible miRNAs, and a novel small RNA in B. mori. The latter, bmo-miR-100-like, was identified using the known miRNA aga-miR-100 as a probe; bmo-miR-100-like was detected by microarray assay and Northern blotting, but its precursor sequences did not fold into a hairpin structure. Among these identified miRNAs, we found 12 pairs of miRNAs and miRNA*s. Northern blotting revealed that some B. mori miRNA genes were expressed only during specific stages, indicating that B. mori miRNA genes (e.g., bmo-miR-277) have developmentally regulated patterns of expression. We identified two miRNA gene clusters in the B. mori genome. bmo-miR-2b, which is found in the gene cluster bmo-miR-2a-1/bmo-miR-2a-1*/bmo-miR-2a-2/bmo-miR-2b/bmo-miR-13a*/bmo-miR-13b, encodes a newly identified member of the mir-2 family. Moreover, we found that methylation can increase the sensitivity of a DNA probe used to detect a miRNA by Northern blotting. Functional analysis revealed that 11 miRNAs may regulate 13 B. mori orthologs of the 25 known Drosophila miRNA-targeted genes according to the functional conservation. We predicted the binding sites on the 1671 3'UTR of B. mori genes; 547 targeted genes, including 986 target sites, were predicted. Of these target sites, 338 had perfect base pairing to the seed region of 43 miRNAs. From the predicted genes, 61 genes, each of them with multiple predicted target sites, should be considered excellent candidates for future functional studies. Biological classification of predicted miRNA targets showed that "binding", "catalytic activity" and "physiological process" were over-represented for the predicted genes. Conclusion: Combining computational predictions with microarray assays, we identified 46 B. mori miRNAs, 13 of which were miRNA*s. We identified a novel small RNA and 21 plausible B. mori miRNAs that could not be located in the available B. mori genome, but which could be detected by microarray. Thirteen and 547 target genes were predicted according to the functional conservation and binding sites, respectively. Identification of miRNAs in B. mori, particularly those that are developmentally regulated, provides a foundation for subsequent functional studies.

Background: The B-BOX (BBX) proteins are the class of zinc-finger transcription factors and can regulate plant growth, development, and endure stress response. In plants, the BBX gene family has been identified in Arabidopsis, rice, and tomato. However, no systematic analysis of BBX genes has been undertaken in grapevine. Results: In this study, 24 grapevine BBX (VvBBX) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Phylogenetic analysis divided VvBBX genes into five subgroups. Numerous cis-acting elements related to plant development, hormone and/or stress responses were identified in the promoter of the VvBBX genes. The tissue-specific expressional dynamics of VvBBX genes demonstrated that VvBBXs might play important role in plant growth and development. The transcript analysis from transcriptome data and qRT-PCR inferred that 11 VvBBX genes were down-regulated in different fruit developmental stages, while three VvBBX genes were up-regulated. It is also speculated that VvBBX genes might be involved in multiple hormone signaling (ABA, ethylene, GA3, and CPPU) as transcriptional regulators to modulate berry development and ripening. VvBBX22 seems to be responsive to multiple hormone signaling, including ABA, ethylene GA3, and CPPU. Some VvBBX genes were strongly induced by Cu, salt, waterlogging, and drought stress treatment. Furthermore, the expression of VvBBX22 proposed its involvement in multiple functions, including leaf senescence, abiotic stress responses, fruit development, and hormone response. Conclusions: Our results will provide the reference for functional studies of BBX gene family, and highlight its functions in grapevine berry development and ripening. The results will help us to better understand the complexity of the BBX gene family in abiotic stress tolerance and provide valuable information for future functional characterization of specific genes in grapevine.

High temperature reduces anthocyanin accumulation in various horticultural plants. However, the molecular mechanisms underlying the high-temperature-induced reduction of anthocyanin in grape (Vitis vinifera) remain poorly understood. In this study, VvMYB44-1 was identified as a transcriptional repressor of anthocyanin biosynthesis in grape berries, and its gene expression was strongly induced by high-temperature treatment. Overexpression of VvMYB44-1 inhibited anthocyanin accumulation in both grape berries and tobacco (Nicotiana tabacum) by repressing the transcription of the anthocyanin biosynthesis genes dihydroflavonol-4-reductase (VvDFR) and UDP-glucose flavonoid-3-O-glucosyltransferase (VvUFGT). Furthermore, the interaction between VvMYB44-1 and VvWDR2 competitively inhibited the formation of the MYB-bHLH-WD40 (MBW) activation complex and weakened the transcriptional activity of the complex, thereby decreasing anthocyanin accumulation. Additionally, VvMYB44-1 facilitated cytokinin (CK) accumulation by upregulating the expression of the CK synthesis gene lonely guy 8 (VvLOG8) and inhibiting the CK degradation gene CK oxidase 4(VvCKX4), thus contributing to CK-mediated anthocyanin inhibition in grape berries. Moreover, the inhibitory effect of VvMYB44-1 on anthocyanin biosynthesis and its downstream target genes was weakened with the deletion of the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, indicating that the EAR motif is indispensable for the inhibitory effect of VvMYB44-1 on anthocyanin biosynthesis in grapes. These results provide insights into the regulatory network of VvMYB44-1 in high-temperature-mediated anthocyanin biosynthesis in grapes.

Growth regulatory factors (GRF) are plant-specific transcription factors that play an important role in plant resistance to stress. This gene family in strawberry has not been investigated previously. In this study, 10 GRF genes were identified in the genome of the diploid woodland strawberry (Fragaria vesca). Chromosome analysis showed that the 10 FvGRF genes were unevenly distributed on five chromosomes. Phylogenetic analysis resolved the FvGRF proteins into five groups. Genes of similar structure were placed in the same group, which was indicative of functional redundance. Whole-genome duplication/segmental duplication and dispersed duplication events effectively promoted expansion of the strawberry GRF gene family. Quantitative reverse transcription-PCR analysis suggested that FvGRF genes played potential roles in the growth and development of vegetative organs. Expression profile analysis revealed that FvGRF3, FvGRF5, and FvGRF7 were up-regulated under low-temperature stress, FvGRF4 and FvGRF9 were up-regulated under high-temperature stress, FvGRF6 and FvGRF8 were up-regulated under drought stress, FvGRF3, FvGRF6, and FvGRF8 were up-regulated under salt stress, FvGRF2, FvGRF7, and FvGRF9 were up-regulated under salicylic acid treatment, and FvGRF3, FvGRF7, FvGRF9, and FvGRF10 were up-regulated under abscisic acid treatment. Promoter analysis indicated that FvGRF genes were involved in plant growth and development and stress response. These results provide a theoretical and empirical foundation for the elucidation of the mechanisms of abiotic stress responses in strawberry.