Designing a synthetic moss genome using GenoDesigner.

The de novo synthesis of genomes has made unprecedented progress and achieved milestones, particularly in bacteria and yeast. However, the process of synthesizing a multicellular plant genome has not progressed at the same pace, due to the complexity of multicellular plant genomes, technical difficulties associated with large genome size and structure, and the intricacies of gene regulation and expression in plants. Here we outline the bottom-up design principles for the de novo synthesis of the Physcomitrium patens (that is, earthmoss) genome. To facilitate international collaboration and accessibility, we have developed and launched a public online design platform called GenoDesigner. This platform offers an intuitive graphical interface enabling users to efficiently manipulate extensive genome sequences, even up to the gigabase level. This tool is poised to greatly expedite the synthesis of the P. patens genome, offering an essential reference and roadmap for the synthesis of plant genomes.

Brassinosteroid regulates stomatal development in etiolated Arabidopsis cotyledons via transcription factors BZR1 and BES1.

Brassinosteroids (BRs) are phytohormones that regulate stomatal development. In this study, we report that BR represses stomatal development in etiolated Arabidopsis (Arabidopsis thaliana) cotyledons via transcription factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS SUPPRESSOR1 (BES1), which directly target MITOGEN-ACTIVATED PROTEIN KINASE KINASE 9 (MKK9) and FAMA, 2 important genes for stomatal development. BZR1/BES1 bind MKK9 and FAMA promoters in vitro and in vivo, and mutation of the BZR1/BES1 binding motif in MKK9/FAMA promoters abolishes their transcription regulation by BZR1/BES1 in plants. Expression of a constitutively active MKK9 (MKK9DD) suppressed overproduction of stomata induced by BR deficiency, while expression of a constitutively inactive MKK9 (MKK9KR) induced high-density stomata in bzr1-1D. In addition, bzr-h, a sextuple mutant of the BZR1 family of proteins, produced overabundant stomata, and the dominant bzr1-1D and bes1-D mutants effectively suppressed the stomata-overproducing phenotype of brassinosteroid insensitive 1-116 (bri1-116) and brassinosteroid insensitive 2-1 (bin2-1). In conclusion, our results revealed important roles of BZR1/BES1 in stomatal development, and their transcriptional regulation of MKK9 and FAMA expression may contribute to BR-regulated stomatal development in etiolated Arabidopsis cotyledons.

A designer synthetic chromosome fragment functions in moss.

Rapid advances in DNA synthesis techniques have enabled the assembly and engineering of viral and microbial genomes, presenting new opportunities for synthetic genomics in multicellular eukaryotic organisms. These organisms, characterized by larger genomes, abundant transposons and extensive epigenetic regulation, pose unique challenges. Here we report the in vivo assembly of chromosomal fragments in the moss Physcomitrium patens, producing phenotypically virtually wild-type lines in which one-third of the coding region of a chromosomal arm is replaced by redesigned, chemically synthesized fragments. By eliminating 55.8% of a 155 kb endogenous chromosomal region, we substantially simplified the genome without discernible phenotypic effects, implying that many transposable elements may minimally impact growth. We also introduced other sequence modifications, such as PCRTag incorporation, gene locus swapping and stop codon substitution. Despite these substantial changes, the complex epigenetic landscape was normally established, albeit with some three-dimensional conformation alterations. The synthesis of a partial multicellular eukaryotic chromosome arm lays the foundation for the synthetic moss genome project (SynMoss) and paves the way for genome synthesis in multicellular organisms.

Brassinosteroids regulate outer ovule integument growth in part via the control of INNER NO OUTER by BRASSINOZOLE-RESISTANT family transcription factors.

Brassinosteroids (BRs) play important roles in regulating plant reproductive processes. BR signaling or BR biosynthesis null mutants do not produce seeds under natural conditions, but the molecular mechanism underlying this infertility is poorly understood. In this study, we report that outer integument growth and embryo sac development were impaired in the ovules of the Arabidopsis thaliana BR receptor null mutant bri1-116. Gene expression and RNA-seq analyses showed that the expression of INNER NO OUTER (INO), an essential regulator of outer integument growth, was significantly reduced in the bri1-116 mutant. Increased INO expression due to overexpression or increased transcriptional activity of BRASSINAZOLE-RESISTANT 1 (BZR1) in the mutant alleviated the outer integument growth defect in bri1-116 ovules, suggesting that BRs regulate outer integument growth partially via BZR1-mediated transcriptional regulation of INO. Meanwhile, INO expression in bzr-h, a null mutant for all BZR1 family genes, was barely detectable; and the outer integument of bzr-h ovules had much more severe growth defects than those of the bri1-116 mutant. Together, our findings establish a new role for BRs in regulating ovule development and suggest that BZR1 family transcription factors might regulate outer integument growth through both BRI1-dependent and BRI1-independent pathways.

Brassinosteroid and Hydrogen Peroxide Interdependently Induce Stomatal Opening by Promoting Guard Cell Starch Degradation.

Starch is the major storage carbohydrate in plants and functions in buffering carbon and energy availability for plant fitness with challenging environmental conditions. The timing and extent of starch degradation appear to be determined by diverse hormonal and environmental signals; however, our understanding of the regulation of starch metabolism is fragmentary. Here, we demonstrate that the phytohormone brassinosteroid (BR) and redox signal hydrogen peroxide (H2O2) induce the breakdown of starch in guard cells, which promotes stomatal opening. The BR-insensitive mutant bri1-116 accumulated high levels of starch in guard cells, impairing stomatal opening in response to light. The gain-of-function mutant bzr1-1D suppressed the starch excess phenotype of bri1-116, thereby promoting stomatal opening. BRASSINAZOLE-RESISTANT1 (BZR1) interacts with the basic leucine zipper transcription factor G-BOX BINDING FACTOR2 (GBF2) to promote the expression of ß-AMYLASE1 (BAM1), which is responsible for starch degradation in guard cells. H2O2 induces BZR1 oxidation, enhancing the interaction between BZR1 and GBF2 to increase BAM1 transcription. Mutations in BAM1 lead to starch accumulation and reduce the effects of BR and H2O2 on stomatal opening. Overall, this study uncovers the critical roles of BR and H2O2 in regulating guard cell starch metabolism and stomatal opening.

BZR1 Family Transcription Factors Function Redundantly and Indispensably in BR Signaling but Exhibit BRI1-Independent Function in Regulating Anther Development in Arabidopsis.

BRASSINAZOLE-RESISTANT 1 family proteins (BZRs) are central transcription factors that govern brassinosteroid (BR)-regulated gene expression and plant growth. However, it is unclear whether there exists a BZR-independent pathway that mediates BR signaling. In this study, we found that disruption of all BZRs in Arabidopsis generated a hextuple mutant (bzr-h) displaying vegetative growth phenotypes that were almost identical to those of the null mutant of three BR receptors, bri1brl1brl3 (bri-t). By RNA sequencing, we found that global gene expression in bzr-h was unaffected by 2 h of BR treatment. The anthers of bzr-h plants were loculeless, but a similar phenotype was not observed in bri-t, suggesting that BZRs have a BR signaling-independent regulatory role in anther development. By real-time PCR and in situ hybridization, we found that the expression of SPOROCYTELESS (SPL), which encodes a transcription factor essential for anther locule development, was barely detectable in bzr-h, suggesting that BZRs regulate locule development by affecting SPL expression. Our findings reveal that BZRs are indispensable transcription factors required for both BR signaling and anther locule development, providing new insight into the molecular mechanisms underlying the microsporogenesis in Arabidopsis.