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.[Keywords: Chlamydomonas; miRNA; mRNA cleavage; small RNA; RNAi; RISC] Supplemental material is available at http://www.genesdev.org.

MADS-box genes form a large family of transcription factors and play important roles in flower development and organ differentiation in plants. In this study, 42 wheat cDNAs encoding putative MADS-box genes were isolated. BLASTX searches and phylogenetic analysis indicated that the cDNAs represented 12 of the 14 MADS-box gene subfamilies. TaAGL14 and TaAGL15 formed a new subfamily along with a rice gene OsMADS32. RT-PCR analysis revealed that these genes had different exprsssion patterns in different organs of different stages. Expression patterns of TaAGL1 and TaAGL29 were also determined using in situ hybridization. TaAGL1 was abundantly expressed in primary root tips and the whole spikelet with more intense labeling at lodicules, paleas and stamens. TaAGL29 was expressed in both the non-reproductive parts (lemma, palea and glumes), and stamens and pistils. Moreover, differential expression patterns of these genes were also observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while others were down-regulated in hybrid as compared to its parents. We concluded that multiple MADS-box genes exist in wheat genome and are expressed in tissue-specific patterns, and might play important roles in wheat growth and development.

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.Flowering time (or heading date) and drought resistance are two major yield traits in crops, especially rice (Oryza sativa). As global climatic change looms, drought has become the biggest abiotic stress to limit crop yields. Breeders have capitalized on naturally occurring genetic variations to improve or maintain crop yield in times or areas of drought by different strategies (Eisenstein, 2013). Manipulation of floral transition has been a promising way to maximize crop yield during dry periods. This strategy has been successful due to extensive identification of genetic loci and elucidation of molecular mechanisms that control flowering time under diverse or unpredictable environments.Heading date in rice is influenced by many environmental cues such as day length (photoperiod), temperature, nutrition, and water availability. Molecular mechanisms that underlie photoperiod regulation of flowering time have already been characterized, probably because day length is more predictable than other environmental factors during seasonal changes. Rice is a facultative short-day plant that flowers earlier in short days (SDs) than in long days (LDs). Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) are two paralogous genes in rice encoding "florigen" molecules expressed in the phloem of leaves and transported to the shoot apical meristem to promote flowering (Tamaki et al., 2007;

SummaryChlamydomonas 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.

Improved soybean cultivars have been adapted to grow at a wide range of latitudes, enabling expansion of cultivation worldwide. However, the genetic basis of this broad adaptation is still not clear. Here, we report the identification of GmPRR3b as a major flowering time regulatory gene that has been selected during domestication and genetic improvement for geographic expansion. Through a genome-wide association study of a diverse soybean landrace panel consisting of 279 accessions, we identified 16 candidate quantitative loci associated with flowering time and maturity time. The strongest signal resides in the known flowering gene E2, verifying the effectiveness of our approach. We detected strong signals associated with both flowering and maturity time in a genomic region containing GmPRR3b. Haplotype analysis revealed that GmPRR3b H6 is the major form of GmPRR3b that has been utilized during recent breeding of modern cultivars. mRNA profiling analysis showed that GmPRR3b H6 displays rhythmic and photoperioddependent expression and is preferentially induced under long-day conditions. Overexpression of GmPRR3b H6 increased main stem node number and yield, while knockout of GmPRR3b H6 using CRISPR/ Cas9 technology delayed growth and the floral transition. GmPRR3b H6 appears to act as a transcriptional repressor of multiple predicted circadian clock genes, including GmCCA1a, which directly upregulates J/GmELF3a to modulate flowering time. The causal SNP (Chr12:5520945) likely endows GmPRR3b H6 a moderate but appropriate level of activity, leading to early flowering and vigorous growth traits preferentially selected during broad adaptation of landraces and improvement of cultivars.

Soybean is an important legume crop that displays the classic shade avoidance syndrome (SAS), including exaggerated stem elongation, which 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 used CRISPR-Cas9-engineered Gmcry mutants to investigate the functions of seven cryptochromes (GmCRYs) in soybean and found that the four GmCRY1s overlap in mediating LBL-induced SAS. Lightactivated GmCRY1s increase the abundance of the bZIP transcription factors STF1 and STF2, which directly upregulate the expression of genes encoding GA2 oxidases to deactivate GA 1 and repress stem elongation. Notably, GmCRY1b overexpression lines displayed multiple agronomic advantages over the wild-type control under both dense planting and intercropping conditions. Our study demonstrates the integration of GmCRY1-mediated signals with the GA metabolic pathway in the regulation of LBL-induced SAS in soybean. It also provides a promising option for breeding lodging-resistant, high-yield soybean cultivars in the future.

The normal functioning of neuroendocrine systems requires that many neuropeptidergic cells change, to alter transmitter identity and concentration, electrical properties, and cellular morphology in response to hormonal cues. During insect metamorphosis, a pulse of circulating steroids, ecdysteroids, governs the dramatic remodeling of larval neurons to serve adult-specific functions. To identify molecular mechanisms underlying metamorphic remodeling, we conducted a neuropeptidergic cell-targeted, gain-of-function genetic screen. We screened 6097 lines. Each line permitted Gal4-regulated transcription of flanking genes. A total of 58 lines, representing 51 loci, showed defects in neuropeptide-mediated developmental transitions (ecdysis or wing expansion) when crossed to the panneuropeptidergic Gal4 driver, 386Y-Gal4. In a secondary screen, we found 29 loci that produced wing expansion defects when crossed to a crustacean cardioactive peptide (CCAP)/bursicon neuron-specific Gal4 driver. At least 14 loci disrupted the formation or maintenance of adult-specific CCAP/bursicon cell projections during metamorphosis. These include components of the insulin and epidermal growth factor signaling pathways, an ecdysteroid-response gene, cabut, and an ubiquitin-specific protease gene, fat facets, with known functions in neuronal development. Several additional genes, including three micro-RNA loci and two factors related to signaling by Myb-like proto-oncogenes, have not previously been implicated in steroid signaling or neuronal remodeling.

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.