Modeling 0.6 million genes for the rational design of functional cis-regulatory variants and de novo design of cis-regulatory sequences.

Rational design of plant cis-regulatory DNA sequences without expert intervention or prior domain knowledge is still a daunting task. Here, we developed PhytoExpr, a deep learning framework capable of predicting both mRNA abundance and plant species using the proximal regulatory sequence as the sole input. PhytoExpr was trained over 17 species representative of major clades of the plant kingdom to enhance its generalizability. Via input perturbation, quantitative functional annotation of the input sequence was achieved at single-nucleotide resolution, revealing an abundance of predicted high-impact nucleotides in conserved noncoding sequences and transcription factor binding sites. Evaluation of maize HapMap3 single-nucleotide polymorphisms (SNPs) by PhytoExpr demonstrates an enrichment of predicted high-impact SNPs in cis-eQTL. Additionally, we provided two algorithms that harnessed the power of PhytoExpr in designing functional cis-regulatory variants, and de novo creation of species-specific cis-regulatory sequences through in silico evolution of random DNA sequences. Our model represents a general and robust approach for functional variant discovery in population genetics and rational design of regulatory sequences for genome editing and synthetic biology.

SHORT ROOT and INDETERMINATE DOMAIN family members govern PIN-FORMED expression to regulate minor vein differentiation in rice.

C3 and C4 grasses directly and indirectly provide the vast majority of calories to the human diet, yet our understanding of the molecular mechanisms driving photosynthetic productivity in grasses is largely unexplored. Ground meristem cells divide to form mesophyll or vascular initial cells early in leaf development in C3 and C4 grasses. Here we define a genetic circuit composed of SHORT ROOT (SHR), INDETERMINATE DOMAIN (IDD), and PIN-FORMED (PIN) family members that specifies vascular identify and ground cell proliferation in leaves of both C3 and C4 grasses. Ectopic expression and loss-of-function mutant studies of SHR paralogs in the C3 plant Oryza sativa (rice) and the C4 plant Setaria viridis (green millet) revealed the roles of these genes in both minor vein formation and ground cell differentiation. Genetic and in vitro studies further suggested that SHR regulates this process through its interactions with IDD12 and 13. We also revealed direct interactions of these IDD proteins with a putative regulatory element within the auxin transporter gene PIN5c. Collectively, these findings indicate that a SHR-IDD regulatory circuit mediates auxin transport by negatively regulating PIN expression to modulate minor vein patterning in the grasses.

Plasma membrane-localized SEM1 protein mediates sugar movement to sink rice tissues.

The translocation of photosynthate carbohydrates, such as sucrose, is critical for plant growth and crop yield. Previous studies have revealed that sugar transporters, plasmodesmata and sieve plates act as important controllers in sucrose loading into and unloading from phloem in the vascular system. However, other pivotal steps for the regulation of sucrose movement remain largely elusive. In this study, characterization of two starch excesses in mesophyll (sem) mutants and dye and sucrose export assays were performed to provide insights into the regulatory networks that drive source-sink relations in rice. Map-based cloning identified two allelic mutations in a gene encoding a GLUCAN SYNTHASE-LIKE (GSL) protein, thus indicating a role for SEM1 in callose biosynthesis. Subcellular localization in rice showed that SEM1 localized to the plasma membrane. In situ expression analysis and GUS staining showed that SEM1 was mainly expressed in vascular phloem cells. Reduced sucrose transport was found in the sem1-1/1-2 mutant, which led to excessive starch accumulation in source leaves and inhibited photosynthesis. Paraffin section and transmission electron microscopy experiments revealed that less-developed vascular cells (VCs) in sem1-1/1-2 potentially disturbed sugar movement. Moreover, dye and sugar trafficking experiments revealed that aberrant VC development was the main reason for the pleiotropic phenotype of sem1-1/1-2. In total, efficient sucrose loading into the phloem benefits from an optional number of VCs with a large vacuole that could act as a buffer holding tank for sucrose passing from the vascular bundle sheath.

OsbHLH92, in the noncanonical brassinosteroid signaling pathway, positively regulates leaf angle and grain weight in rice.

Modifications of plant architecture can increase planting density, regulate photosynthesis, and improve crop yields. Many basic helix-loop-helix (bHLH) transcription factors participate in the brassinosteroid (BR) signaling pathway and are critical for plant architecture morphogenesis in rice. However, the number of identified bHLH genes suitable for improving production value is still limited. In this study, we cloned Lam1, encoding the typical bHLH transcription factor OsbHLH92. OsbHLH92 knockout (KO) lines exhibit erect leaves. Decreases in the number and size of parenchyma cell layers on the adaxial side of the lamina joint in KO lines were the main reason for the decreased leaf angle. Genetic experiments verify that OsBU1 and its homologs are downstream of OsbHLH92, which is involved in the noncanonical RGA1-mediated BR signaling pathway. OsbHLH91, an OsbHLH92 homolog, plays both conserved and differentiated roles relative to OsbHLH92. Notably, OsbHLH92-KO lines show erect leaves without the acquisition of adverse agronomic traits. Moreover, by driving a specific panicle promoter, OsbHLH92 can greatly increase productivity by at least 10%. This study identifies new components of the BR signaling pathway, demonstrates the importance of OsbHLH92 in improving planting density and crop productivity, and broadens our knowledge of typical and atypical bHLH family members in rice.

A Subsidiary Cell-Localized Glucose Transporter Promotes Stomatal Conductance and Photosynthesis.

It has long been recognized that stomatal movement modulates CO2 availability and as a consequence the photosynthetic rate of plants, and that this process is feedback-regulated by photoassimilates. However, the genetic components and mechanisms underlying this regulatory loop remain poorly understood, especially in monocot crop species. Here, we report the cloning and functional characterization of a maize (Zea mays) mutant named closed stomata1 (cst1). Map-based cloning of cst1 followed by confirmation with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 system identified the causal mutation in a Clade I Sugars Will Eventually be Exported Transporters (SWEET) family gene, which leads to the E81K mutation in the CST1 protein. CST1 encodes a functional glucose transporter expressed in subsidiary cells, and the E81K mutation strongly impairs the oligomerization and glucose transporter activity of CST1. Mutation of CST1 results in reduced stomatal opening, carbon starvation, and early senescence in leaves, suggesting that CST1 functions as a positive regulator of stomatal opening. Moreover, CST1 expression is induced by carbon starvation and suppressed by photoassimilate accumulation. Our study thus defines CST1 as a missing link in the feedback-regulation of stomatal movement and photosynthesis by photoassimilates in maize.

HD-ZIP IV gene Roc8 regulates the size of bulliform cells and lignin content in rice.

The morphology of bulliform cells located on the upper epidermis of leaves is one of the most important cell structures affecting leaf shape. Although many mechanisms regulating the development of bulliform cells have been reported, the fine regulatory mechanisms governing this process have rarely been described. To identify novel components regulating rice leaf morphology, a mutant showing a constitutively rolling phenotype from the seedling stage to flowering, known as crm1-D, was selected for further analysis. Anatomical analyses in crm1-D were attributable to the size reduction of bulliform cells. The crm1-D was controlled by a single dominant nuclear gene. Map-based cloning revealed that Roc8, an HD zipper class IV family member, was responsible for the crm1-D phenotype. Notably, the 50-bp sequence in the 3'-untranslated region (3'-UTR) of the Roc8 gene represses Roc8 at the translational level. Moreover, the roc8 knockdown lines notably increased the size of bulliform cells. A series of assays revealed that Roc8 negatively regulates the size of bulliform cells. Unexpectedly, Roc8 was also observed to positively mediate lignin biosynthesis without incurring a production penalty. The above results show that Roc8 may have a practical application in cultivating materials with high photosynthetic efficiency and low lignin content.

RLM1, Encoding an R2R3 MYB Transcription Factor, Regulates the Development of Secondary Cell Wall in Rice.

Leaf morphology is an important component of rice ideal plant type. To date, many regulatory genes influencing leaf morphology in rice have been cloned, and their underlying molecular regulatory mechanism has been preliminarily clarified. However, the fine regulation relationship of leaf morphogenesis and plant type remains largely elusive. In this study, a rolling-leaf mutant, named rlm1-D, was obtained and controlled by a pair of dominant nuclear genes. Cytological observations revealed that the rlm1 was mainly caused by abnormal deposition of secondary cell walls. Molecular evidence showed ectopic expression of a MYB-type transcription factor LOC_Os05g46610 was responsible for the phenotype of rlm1-D. A series of experiments, including the transcription factor-centered technology, DNA-binding assay, and electrophoretic mobility shift assay, verified that RLM1 can bind to the promoter of OsCAD2, a key gene responsible for lignin biosynthesis in rice. An interacting partner of RLM1, OsMAPK10, was identified. Multiple biochemical assays confirmed that OsMAPK10 interacted with RLM1. OsMAPK10 positively regulated the lignin content in the leaves and stems of rice. Moreover, OsMAPK10 contributes to RLM1 activation of downstream target genes. In particular, RLM1 is exclusively expressed in the stems at the mature plant stage. The yield of RLM1 knockdown lines increased by over 11% without other adverse agricultural trait penalties, indicating great practical application value. A MAPK-MYB-OsCAD2 genetic regulatory network controlling SCW was proposed, providing a theoretical significance and practical value for shaping the ideal plant type and improving rice yield.

OsRELA Regulates Leaf Inclination by Repressing the Transcriptional Activity of OsLIC in Rice.

Leaf angle is one of the most important agronomic traits in rice, and changes in leaf angle can alter plant architecture to affect photosynthetic efficiency and thus determine grain yield. Therefore, it is important to identify key genes controlling leaf angle and elucidate the molecular mechanisms to improve rice yield. We obtained a mutant rela (regulator of leaf angle) with reduced leaf angle in rice by EMS mutagenesis, and map-based cloning revealed that OsRELA encodes a protein of unknown function. Coincidentally, DENSE AND ERECT PANICLE 2 (DEP2) was reported in a previous study with the same gene locus. RNA-seq analysis revealed that OsRELA is involved in regulating the expression of ILI and Expansin family genes. Biochemical and genetic analyses revealed that OsRELA is able to interact with OsLIC, a negative regulator of BR signaling, through its conserved C-terminal domain, which is essential for OsRELA function in rice. The binding of OsRELA can activate the expression of downstream genes repressed by OsLIC, such as OsILI1, a positive regulator of leaf inclination in rice. Therefore, our results suggest that OsRELA can act as a transcriptional regulator and is involved in the regulation of leaf inclination by regulating the transcriptional activity of OsLIC.

Transcriptome profiling revealed novel transcriptional regulators in maize responses to Ostrinia furnacalis and jasmonic acid.

Chewing insects cause severe yield losses in crop production worldwide. Crop plants counteract chewing insects by transcriptionally promoting a repertoire of defense gene products that are either toxic to, or attractive to the natural enemies of, pest insects. However, the complexity of the transcriptional reprogramming in plant defense response against chewing insects is still not well understood. In this study, the genome-wide early responses in maize seedlings to Asian corn borer (ACB, Ostrinia furnacalis) and also to jasmonic acid(JA), the pivotal phytohormone controlling plant defense response against herbivory, were transcriptionally profiled by RNA-Seq. Clustering of differentially expressed genes (DEGs) along with functional enrichment analysis revealed important biological processes regulated in response to ACB infestation and/or jasmonic acid. Moreover, DEGs with distinct expression patterns were differentially enriched with diverse families of cis-elements on their promoters. Multiple inventories of differentially expressed transcription factors (DETFs) in each DEG group were also analyzed. A transient expression assay using transfected maize protoplastswas established to examine the potential roles of DETFs in maize defense response and JA signaling, and this was used to show that ZmNAC60, an ACB- and JA-inducible DETF, represented a novel positive regulator of JA and defense pathway genes. This study provided a comprehensive transcriptional picture for the early dynamics of maize defense responses and JA signaling, and the identification of DETFs offered potential targets for further functional genomics investigation of master regulators in maize defense responses against herbivory.