Aigen Fu

Juglans regia cv. Zijing, known as ‘Five-purple walnut’ exhibits distinct purple phenotypes in leaves, husks, flowers, branches, and seed coats. Despite its high horticultural value and stress resistance, the genetic basis of its unusual anthocyanin accumulation and the regulatory mechanisms underlying its purple phenotypes remain poorly understood. Here, we report chromosome-level genomes for ZJ and wild-type ‘Lvling’ (LL) walnuts, enabling comparative insights into genes regulating anthocyanin accumulation in plants. We identified genetic variations of JrATs, JrGHs, JrGH1s, and JrNLRs as unique to ZJ. By integrating transcriptomic and metabolomic analyses, we identified 590 differentially expressed genes (DEGs) and 118 differentially accumulated metabolites (DAMs) between purple and wild-type tissues. Consistent with leaf and husk data, enzymes JrANS54, JrANS102, JrC4H122, JrUFGT46, and transcription factors (TFs), particularly the MYB-bHLH-WD40 were expressed highly in ZJ leaves and husks. Multi-omics integration converged on JrMYB216 and JrMYB85 as important TFs regulating purple colorations in ZJ. Functional validation through stable Arabidopsis transformation and transient walnut husk assays confirmed that JrMYB216 and JrMYB85 form MYB-bHLH-WD40 complex with JrbHLH42 and JrWD40–168. These complexes synergistically activated structural genes downstream of the anthocyanin biosynthetic pathway, promoting proanthocyanidins and delphinidin derivative accumulation, thereby driving the purple phenotype. Notably, JrMYB216 contributed more significantly to anthocyanin accumulation than JrMYB85, with its enhanced regulatory activity potentially linked to a 23 bp deletion in the second intron. This study systematically elucidates the regulatory network governing anthocyanin mediated purple phenotyping in walnut, providing novel theoretical insights into the molecular mechanisms underlying purple coloration traits in perennial woody plants.

SUMMARY: Hydrogen (H ₂ ) photoproduction by green microalgae offers a promising renewable energy strategy, yet its efficiency is constrained by the oxygen sensitivity of hydrogenases and the availability of photosynthetic electrons produced by photosystem II (PSII). Conventional approaches such as sulfur deprivation to induce H ₂ photoproduction inevitably lead to downregulation of PSII and thereafter restriction of the sufficient electron supply to hydrogenases. Here, we show that the thylakoid membrane phosphatase TEF8 (Thylakoid Enriched Fraction 8) acts as a negative regulator of H ₂ photoproduction in Chlamydomonas reinhardtii under sulfur‐deprivation conditions. Compared with wild type, the tef8 knockout mutant exhibits significantly enhanced H ₂ evolution and increased stability of PSII supercomplexes when H ₂ photoproduction was induced. Further biochemical analyses reveal that TEF8 is involved in the dephosphorylation of PSII subunits, promoting the degradation of PSII core complex under sulfur deprivation. Loss of TEF8 function impairs dephosphorylation of D2 and CP29, thereby stabilizing PSII supercomplexes and maintaining higher electron flux to hydrogenases under sulfur‐deprivation conditions. These findings identify TEF8 as a regulatory protein that fine‐tunes PSII activity and photosynthetic electron flow, thereby modulating hydrogen photoproduction efficiency.

Humic acid (HA) is known to improve phosphorus (P) availability in agricultural soils. Yet, the underlying mechanisms by which it influences the microbial community and subsequent P turnover remain unclear. In this study, a pot experiment was conducted using soil from a three-year Salvia miltiorrhiza ( S. miltiorrhiza ) cultivation system, in which the soil was amended with three HA concentrations (T1: 100-fold dilution, T2: 200-fold, T3: 400-fold) alongside an untreated control (CK). We determined P uptake by S. miltiorrhiza , soil P fractions, phosphatase activities, along with high-throughput sequencing of the microbial communities to specifically target those associated with P transformation. The results showed that HA application significantly enhanced root P uptake, with increases of 68.59% and 91.05% under T2 and T3, respectively. Consequently, soil Olsen-P content decreased by 19.19% and 15.20%, respectively, consistent with the depletion of available P under enhanced plant uptake. Soil P fractionation further revealed that HA application decreased inorganic P by 20.91% and 32.29%, respectively, under T2 and T3 treatments. Specifically, H ₂ O-P decreased by 62.1% and 73.61%, and NaHCO ₃ -Pi decreased by 53.21% and 50.48%, respectively, under the T2 and T3 treatments. In parallel, acid phosphatase activity was increased by 68.72% and 66.67% under the T2 and T3 treatments compared to CK. Comparative high-throughput sequencing between T2 and CK revealed that HA application enriched key microbial genera associated with P cycling, including Sphingomonas , Nitrospira , Ferruginibacter , and Hyphomicrobium . Collectively, these findings suggest that HA may promote P mobilization and mineralization through association with microbial communities, thereby potentially enhancing P bioavailability and plant uptake. These findings offer new perspectives on the associations between HA application, microbial community shifts, and P use efficiency, suggesting a potential approach that merits further evaluation for P management in agricultural systems.

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.

Key message Two Restorer-of-fertility loci, Rf-m1 and Rf-m2, co-condition fertility restoration in ZD-type CMS soybean though complex genetic interactions in both gametophytic and sporophytic manners. Abstract In soybean (Glycine max), the genetic mechanisms underlying fertility restoration of ZD-type cytoplasmic male sterility (CMS-ZD) are not fully understood. By crossing the CMS-ZD line W931A with its strong restorer WR016, we elucidated the inheritance patterns of fertility restoration. We discovered that restoration by WR016 is primarily governed by two Restorer-of-fertility (Rf) loci, Rf-m1 and Rf-m2, located within ~ 220 kb on chromosome 16 and ~ 117 kb on chromosome 9, respectively. They exhibited two distinctive inheritance modes across genotype combinations. In plants with the genotype of -/rf-m1 -/rf-m2, such as F₁ hybrids, gametophytic inheritance mode is predominant. In this case, Rf-m1 in haploid tissue is essential for pollen fertility restoration, and Rf-m2 is a modifier to enhance the effect. Correspondingly, plants displayed low pollen staining rates (sterile to ~ 50% depending on genotype). Conversely, in plants with one locus present as the homozygous Rf/Rf genotype, sporophytic inheritance mode is predominant. Plants exhibited high pollen staining rates (mostly > 80%), and the segregation of heterozygous loci in the next generation followed the Mendelian ratio. Notably, alleles rf-m1 and especially rf-m2 appear to suppress fertility. Further experiments revealed the best candidates of Rf-m1 and Rf-m2 are Glyma.16G139800 and Glyma.09G171200, encoding mitochondrial pentatricopeptide repeat (PPR) proteins GmPPR567 and GmPPR497. Haplotype analysis revealed that lines carrying homozygous Rf /Rf or rf /rf at both loci are rare among domesticated soybean germplasms. Using locus-specific markers and in silico haplotype screening, we identified two effective restorers and one maintainer line in elite germplasms, facilitating hybrid breeding in soybean.

Cyclophilin 38 (CYP38) plays a crucial role in the assembly and stability of photosystem II (PSII), but its molecular mechanism remains unclear. In this study, we identified thylakoid lumen protein 7.6 (TLP7.6) as an in vivo interactor of CYP38. Under normal growth conditions, the tlp7.6 single mutant exhibited no significant phenotypic differences compared to wild-type Col-0. However, the cyp38-2/tlp7.6-1 double mutant displayed severe developmental defects, including stunted growth, delayed flowering, yellowish leaf, short primary roots, abnormal chloroplast ultrastructure, and reduced biomass, which were more pronounced than those in either the tlp7.6 or cyp38-2 single mutant. Photosynthetic analysis revealed that PSII capacities in cyp38-2/tlp7.6-1 and cyp38-2 mutants were significantly reduced, consistent with their slow-growth phenotype. Blue native PAGE analysis demonstrated a substantial reduction in PSII supercomplexes and light-harvesting complex II (LHCII) in cyp38-2/tlp7.6-1, while PSII monomer (PSII-M) were significantly increased. Immunoblotting and two-dimensional gel electrophoresis further confirmed decreased levels of key components of PSII, PSI, and ATPase subunits in the double mutant. Altogether, these results highlight the role of TLP7.6 as an assistive factor in CYP38-mediated PSII assembly, and provide insights into thylakoid lumen protein function in photosynthesis.

SUMMARY: The sophisticated regulation of state transition is required to maintain optimal photosynthetic performance under fluctuating light condition, through balancing the absorbed light energy between photosystem II and photosystem I. This exquisite process incorporates phosphorylation and dephosphorylation of light‐harvesting complexes and PSII core subunits, accomplished by thylakoid membrane‐localized kinases and phosphatases that have not been fully identified. In this study, one Chlamydomonas high light response gene, THYLAKOID ENRICHED FRACTION 8 (TEF8), was characterized. The Chlamydomonas tef8 mutant showed high light sensitivity and defective state transition. The enzymatic activity assays showed that TEF8 is a bona fide phosphatase localized in thylakoid membranes. Biochemical assays, including BN‐PAGE, pull‐down, and phosphopeptide mass spectrometry, proved that TEF8 associates with photosystem II and is involved in the dephosphorylation of D2 and CP29 subunits during state 2 to state 1 transition. Taken together, our results identified TEF8 as a thylakoid phosphatase with multiple dephosphorylation targets on photosystem II, and provide new insight into the regulatory mechanism of state transition and high light resistance in Chlamydomonas.

Summary: Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well‐established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored. Using LC–MS/MS‐based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots. AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the ‘actin cytoskeleton‐26S proteasome‐YUC8‐auxin’ axis that controls auxin levels. Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome.