Tengyue Zhang

BHLH proteins regulate plant development, hormone responses, and stress adaptation; however, the precise functions of most bHLHs remain unclear. In this study, we explored the functions and mechanism of a bHLH gene, IBH1-LIKE 1 (IBL1), in Arabidopsis. IBL1 negatively controlled root growth, hypocotyl elongation, and leaf expansion, as indicated by larger or longer cells in ibl1 compared with Col-0 in the early vegetative process. IBL1 promoted photomorphogenesis and participated in various hormone pathways. In later vegetative stages, ibl1 plants were smaller and weaker than Col-0. During the reproductive stage, the deficiency of IBL1 led to earlier bolting and unhealthy shoots but did not obviously impair gametogenesis and embryogenesis. Longer pistils occurred in ibl1 plants, while stamens exhibited normal length, indicating that IBL1 restricted pistil cell elongation. The unsynchronized sexual organs led to substantial incomplete pollination and vacant seats in siliques. Transcriptomic analysis implied that IBL1 functions via affecting the expression of cell expansion-related genes. Yeast two-hybrid, bimolecular fluorescence complementation, in vitro protein pull-down, electrophoretic mobility shift assays, and dual luciferase assays indicated that IBL1 interacts with other bHLHs, including PRE5, HBI1, and CIL2. These bHLH proteins could constitute a tri-antagonistic system to regulate plant cell elongation/expansion. In a proposed model, IBL1 and PRE5 antagonistically regulate the activity of HBI1/CIL2 to control target gene expression and eventually fine-tune cell elongation/expansion. This study reveals physiological roles and the working mode of IBL1 and provides insights into the regulation of plant growth and development.

Summary: Plastids' function and integrity require quality control of their proteins, and their failure often leads to impairments in plant growth and development. In most angiosperms, plastids propagation exhibits a maternal inheritance pattern. Despite the identification of mechanisms and events related to embryo sac development, the potential role of plastids in controlling female gametophyte (FG) development remains elusive. To characterize the function of a plastid specific cochaperonin (Cpn20) in Arabidopsis, morphological and histological analyses were conducted, combining genetic and cytological analysis with cell‐specific fluorescent labeling detection; RNA sequencing and proteome quantitation were performed. We observed that a weak Cpn20 allele (cpn20‐a) showed pleiotropic defects of chlorosis, dwarfism, and delayed flowering, while the knockout cpn20‐b allele was lethal with abortive ovules in siliques. The embryo sac with the Cpn20 defect was arrested at the single‐cell functional megaspore stage with no observation of plastids. Transcriptome and proteome profiling indicated that Cpn20 malfunction affects chloroplast‐involved functions during the vegetative stage, while disturbing proplastid biogenesis and cell division during the reproductive stage. We rationalized that Cpn20 deficiency leads to a failure of plastid integrity and biogenesis. Our results reveal that Cpn20 plays a key role in plant growth and FG development via its indispensable function for plastids.

Photosystem I (PSI) is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Co-immunoprecipitation, mass spectrometry, and yeast two-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight (LMW) PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-LHCI supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.