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.

Enhancer trap systems have been demonstrated to increase the effectiveness of gene identification in rice. In this study, a chlorophyll-deficient mutant, named oscdm1, was screened and characterized in detail from a T-DNA enhancer-tagged population. The oscdm1 plants were different from other chlorophyll-deficient mutants; they produced chlorotic leaves at the third leaf stage, which gradually died with further growth of the plants. However, the oscdm1 plants were able to survive exposure to elevated CO2 levels, similar to photorespiratory mutants. An analysis of the T-DNA flanking sequence in the oscdm1 plants showed that the T-DNA was inserted into the promoter region of a serine hydroxymethyltransferase gene. OsSHMT1 is a key enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for the interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Full-length OsSHMT1 complemented the oscdm1 phenotype, and the downregulation of OsSHMT1 in wild-type plants by RNA interference produced plants that mimicked the oscdm1 phenotype. GUS assays and quantitative PCR revealed the preferential expression of OsSHMT1 in young leaves. TEM revealed serious damage to the thylakoid membrane in oscdm1 chloroplasts. The oscdm1 plants showed more extensive damage than wild type using an IMAGING-PAM fluorometer, especially under high light intensities. OsSHMT1-GFP localized exclusively to mitochondria. Further analysis revealed that the H2O2 content in the oscdm1 plants was twice that in wild type at the fourth leaf stage. This suggests that the thylakoid membrane damage observed in the oscdm1 plants was caused by excessive H2O2. Interestingly, OsSHMT1-overexpressing plants exhibited increased photosynthetic efficiency and improved plant productivity. These results lay the foundation for further study of the OsSHMT1 gene and will help illuminate the functional role of OsSHMT1 in photorespiration in rice.

Photorespiration, often considered as a wasteful process, is a key target for bioengineering to improve crop yields. Several photorespiratory bypasses have been designed to efficiently metabolize 2-phosphoglycolate and increase the CO2 concentration in chloroplasts, thereby reducing photorespiration. However, the suppression of primary nitrate assimilation remains an issue when photorespiration is inhibited. In this study, we designed a carbon and nitrogen metabolism–coupled photorespiratory bypass, termed the GCBG bypass, in rice (Oryza sativa) chloroplasts. Our results demonstrated efficient assembly and expression of the GCBG bypass in rice chloroplasts, which affected the levels of typical metabolites and their derivatives of natural photorespiration and enhanced the photosynthetic efficiency. Metabolomic analyses revealed that oxaloacetate, produced from glycolate in chloroplasts, positively impacted amino acid synthesis, energy metabolism, and sugar synthesis. The engineered GCBG plants showed an average yield increase of 19.0% (17.8% to 20.2%) compared with wild-type plants under natural growth conditions, alongside improved nitrogen uptake, which compensated for 44.1% of yield losses under nitrogen-limited conditions. In summary, the GCBG bypass substantially improved the photosynthetic efficiency, biomass, and yield in rice by integrating carbon and nitrogen metabolism. This study introduces a strategy for engineering high-yielding rice or other crops with improved photosynthetic efficiency and nitrogen uptake.

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 SHR (SHORT ROOT), IDD (INDETERMINATE DOMAIN), and PIN (PIN-FORMED) 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.