Endogenous small RNAs function in RNA interference (RNAi) pathways to control gene expression through mRNA cleavage, translational repression, or chromatin modification. Plants and animals contain many microRNAs (miRNAs) that play vital roles in development, including helping to specify cell type and tissue identity. To date, no miRNAs have been reported in unicellular organisms. Here we show that Chlamydomonas reinhardtii, a unicellular green alga, encodes many miRNAs. We also show that a Chlamydomonas miRNA can direct the cleavage of its target mRNA in vivo and in vitro. We further show that the expression of some miRNAs/Candidates increases or decreases during Chlamydomonas gametogenesis. In addition to miRNAs, Chlamydomonas harbors other types of small RNAs including phased small interfering RNAs (siRNAs) that are reminiscent of plant trans-acting siRNAs, as well as siRNAs originating from protein-coding genes and transposons. Our findings suggest that the miRNA pathway and some siRNA pathways are ancient mechanisms of gene regulation that evolved prior to the emergence of multicellularity.

Summary: Chlamydomonas reinhardtii is a unicellular green alga. It is a model system for studying functions of the chloroplast, basal body and flagella. The completion of the Chlamydomonas genome sequence makes it possible to use reverse genetic approaches in this organism. Chlamydomonas contains a set of endogenous microRNAs (miRNAs) that down‐regulate their target gene expression through mRNA cleavage. Here we developed an artificial miRNA‐based strategy to knock down gene expression in Chlamydomonas. Using an endogenous Chlamydomonas miRNA precursor as the backbone, we constructed two artificial miRNAs (amiRNAs) targeting the MAA7 and RBCS1/2 genes, respectively. When overexpressed, these two amiRNAs could cleave their respective targets precisely at the predicted sites, resulting in greatly decreased accumulation of MAA7 and RBCS1/2 transcripts and expected mutant phenotypes. We further showed that the two amiRNAs could be produced simultaneously from a dimeric amiRNA precursor. We anticipate that the amiRNA technology developed in this study will be very useful in assessing the functions of individual genes and in genome‐wide approaches.

Soybean as an important legume crop displays classic shade avoidance syndrome (SAS) including exaggerated stem elongation that leads to lodging and yield reduction under density farming conditions. Here we compared the effects of two shade signals, low red light to far-red light ratio (R:FR) and low blue light (LBL), on soybean status and revealed that LBL predominantly induces excessive stem elongation. We inspected the functions of seven cryptochromes (GmCRYs) in soybean utilizing the CRISPR/Cas9 engineered Gmcry mutants. The results show that the four GmCRY1s are overlapping in mediating LBL-induced SAS in soybean. Light-activated GmCRY1s increase the abundance of bZIP transcription factors STF1 and STF2, which directly upregulate the expression of genes encoding GA2-oxidases to deactivate GA1 and repress stem elongation. Notably, the GmCRY1b overexpression lines displayed multiple agronomic advantages over the wild type control under both dense planting and intercropping conditions. Taken together, our study integrates the GmCRY1s-mediated signals with the GA metabolic pathway in regulating LBL-induced SAS in soybean. It provides a promising option to breed lodging-resistant and high yield soybean cultivars in the future.

Plant height is a major trait affecting yield potential in rice. Using a large-scale hybrid transcription factor approach, we identified the novel MYB-like transcription factor OsMPH1 (MYB-like gene of Plant Height 1), which is involved in the regulation of plant height in rice. Overexpression of OsMPH1 leads to increases of plant height and grain yield in rice, while knockdown of OsMPH1 leads to the opposite phenotypes. Microscopy of longitudinal stem sections indicated that a change in internode cell length resulted in the change in plant height. RNA sequencing (RNA-seq) analysis of transgenic rice lines showed that multiple genes related to cell elongation and cell wall synthesis, which are associated with plant height and yield phenotypes, exhibited an altered expression profile. These results imply that OsMPH1 might be involved in specific recognition and signal transduction processes related to plant height and yield formation, providing further insights into the mechanisms underlying the regulation of plant height and providing a candidate gene for the efficient improvement of rice yield.

The molecular mechanisms underlying photoperiod or temperature control of flowering time have been recently elucidated, but how plants regulate flowering time in response to other external factors, such as water availability, remains poorly understood. Using a large-scale Hybrid Transcription Factor approach, we identified a bZIP transcriptional factor, O. sativa ABA responsive element binding factor 1 (OsABF1), which acts as a suppressor of floral transition in a photoperiod-independent manner. Simultaneous knockdown of both OsABF1 and its closest homologous gene, OsbZIP40, in rice (Oryza sativa) by RNA interference results in a significantly earlier flowering phenotype. Molecular and genetic analyses demonstrate that a drought regime enhances expression of the OsABF1 gene, which indirectly suppresses expression of the Early heading date 1 (Ehd1) gene that encodes a key activator of rice flowering. Furthermore, we identified a drought-inducible gene named OsWRKY104 that is under the direct regulation of OsABF1. Overexpression of OsWRKY104 can suppress Ehd1 expression and confers a later flowering phenotype in rice. Together, these findings reveal a novel pathway by which rice modulates heading date in response to the change of ambient water availability.