Knockout of miR396 genes increases seed size and yield in soybean.

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.

Transcription factors WOX11 and LBD16 function with histone demethylase JMJ706 to control crown root development in rice.

Crown roots are the main components of root systems in cereals. Elucidating the mechanisms of crown root formation is instrumental for improving nutrient absorption, stress tolerance, and yield in cereal crops. Several members of the WUSCHEL-related homeobox (WOX) and lateral organ boundaries domain (LBD) transcription factor families play essential roles in controlling crown root development in rice (Oryza sativa). However, the functional relationships among these transcription factors in regulating genes involved in crown root development remain unclear. Here, we identified LBD16 as an additional regulator of rice crown root development. We showed that LBD16 is a direct downstream target of WOX11, a key crown root development regulator in rice. Our results indicated that WOX11 enhances LBD16 transcription by binding to its promoter and recruiting its interaction partner JMJ706, a demethylase that removes histone H3 lysine 9 dimethylation (H3K9me2) from the LBD16 locus. In addition, we established that LBD16 interacts with WOX11, thereby impairing JMJ706-WOX11 complex formation and repressing its own transcriptional activity. Together, our results reveal a feedback system regulating genes that orchestrate crown root development in rice, in which LBD16 acts as a molecular rheostat.

WOX11 and CRL1 act synergistically to promote crown root development by maintaining cytokinin homeostasis in rice.

Crown root (CR) morphogenesis is critical for normal growth and nutrition absorption in cereals. In rice, WUSCHEL-RELATED HOMEOBOX11 (WOX11) and CROWN ROOTLESS1 (CRL1) play vital roles in controlling CR development. Despite their importance, whether and how the two regulators coordinate CR formation remains unclear. Electrophoretic mobility shift assays, transient expression, and chromatin immunoprecipitation qPCR suggested that WOX11 and CRL1 directly bind to OsCKX4 to regulate its expression during CR development. CRL1 enhances OsCKX4 activation through direct interaction with WOX11 at root emergence and elongation stages. Genetic dissection showed that the wox11/crl1 double mutant exhibits a more severe root phenotype. OsCKX4 knockout plants generated by CRISPR/Cas9 exhibited fewer CRs and higher cytokinin levels in the root meristem. Increased expression of OsCKX4 could partially complement the CR phenotypes of both crl1 and wox11 mutants. Furthermore, cytokinin can promote WOX11 protein accumulation in the root meristem. Together, these findings show that cytokinin accumulation is tightly regulated by the WOX11-CRL1 complex during CR elongation by counteracting the negative regulatory effects of cytokinin on root development. Importantly, these results reveal an intrinsic link between WOX11 protein accumulation and cytokinin to maintain CR growth.

A Non-Canonical MITE in the WOX11 Promoter Is Associated with Robust Crown Root development in Rice.

The formation of tissues and organs in multicellular organisms is tightly controlled by transcriptional programs determined by temporal and spatial patterns of gene expression. As an important regulator of rice crown root development, WOX11 is essential for crown root formation and its transcript level is positively correlated with crown root biomass. However, how WOX11 is regulated during crown root primordium emergence and outgrowth still remains unknown. In this study, variations of the WOX11 genomic sequence were analyzed, and the highest genetic diversity was found within its promoter, which contained a non-canonical miniature inverted-repeat transposable element (ncMITE) sequence. Analysis of the WOX11 promoter-driven reporter gene GUS (ß-glucuronidase) transgenic plants pWOX11(ncMITE+):GUS and pWOX11(ncMITE-):GUS uncovered higher GUS expression levels in crown roots of pWOX11(ncMITE+):GUS plants. Furthermore, pWOX11(ncMITE+):WOX11-FLAG in wox11 background could complement the crown root number and length compared to those of the wild type, while pWOX11(ncMITE-):WOX11-FLAG could not. These results suggested that the ncMITE was positively associated with WOX11 transcripts in rice crown roots. In addition, DNA methylation nearby the ncMITE region attenuated the activation effect of the ncMITE on WOX11 expression, which might also be the cause conferred to the root-specific expression of WOX11. This work provides novel insight into WOX11 expression regulation and reveals a promising target for genetic improvement of root architecture in rice.

New insight into comprehensive analysis of INDETERMINATE DOMAIN (IDD) gene family in rice.

The INDETERMINATE DOMAIN (IDD) transcription factor (TF), as a family of plant-specific zinc-finger proteins, regulates a variety of development processes and abiotic stresses in plants. IDD genes have been identified and characterized in other plants, however, the rice IDD family genes have not been investigated at genome-wide. In this study, 15 OsIDD genes were identified in rice genome and phylogenetically classified into two groups. Conserved motifs and potential interaction protein analysis about OsIDD proteins were carried out. Exon-intron structures, cis-acting elements and expression profiles of OsIDD genes were also examined. Exon-intron structures analysis revealed that overall structures of OsIDD genes were relatively conserved although they contained different numbers of introns. Cis-acting elements analysis suggested that most OsIDD gene transcripts could be induced by various abiotic stresses and phytohormones. The expression patterns of OsIDD genes were detected by qRT-PCR under cold and drought conditions, and by exogenous auxin (2,4-D), gibberellin (GA3), and abscisic acid (ABA) treatments, respectively. The results showed that the OsIDDs might play essential roles under abiotic stresses and hormone responses. Distinct expression profiles in tissues/organs suggested that OsIDDs might be involved in different development processes in rice. More interestingly, the prediction of protein-protein interactions (PPIs) revealed OsIDDs could cooperate with some histone modifiers. Yeast two-hybrid assays were performed and confirmed it. Collectively, these results provide a foundation for further elucidation on the molecular mechanisms of OsIDD genes and advance our understanding of their biological function in rice.