Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family

The fibrous root system in cereals comprises primarily adventitious roots （ARs）, which play important roles in nutrient and water uptake. Current knowledge regarding the molecular mechanism underlying AR development is still limited. We report here the isolation of four rice （Oryza sativa L.） mutants, from different genetic backgrounds, all of which were defective in AR formation. These mutants exhibited reduced numbers of lateral roots （LRs） and partial loss of gravitropism. The mutants also displayed enhanced sensitivity to N-l-naphthylphthalamic acid, an inhibitor of polar auxin transport （PAT）, indicating that the mutations affected auxin transport. Positional cloning using one of the four mutants revealed that it was caused by loss-of-function of a guanine nucleotide exchange factor for ADP- ribosylation factor （OsGNOM1）. RT-PCR and analysis of promoter：：GUS transgenic plants showed that OsGNOM1 is expressed in AR primordia, vascular tissues, LRs, root tips, leaves, anthers and lemma veins, with a distribution pattern similar to that of auxin. In addition, the expressions of OsPIN2, OsPIN5b and OsPIN9 were altered in the mutants. Taken together, these findings indicate that OsGNOM1 affects the formation of ARs through regulating PAT.

High-resolution anatomical and spatial transcriptome analyses reveal two types of meristematic cell pools within the secondary vascular tissue of poplar stem

The secondary vascular tissue emanating from meristems is central to understanding how vascular plants such as forest trees evolve,grow,and regulate secondary radial growth.However,the overall molecular characterization of meristem origins and developmental trajectories from primary to secondary vascular tissues in woody tree stems is technically challenging.In this study,we combined high-resolution anatomic analysis with a spatial transcriptome(ST)technique to define features of meristematic cells in a developmental gradient from primary to secondary vascular tissues in poplar stems.The tissue-specific gene expression of meristems and derived vascular tissue types were accordingly mapped to specific anatomical domains.Pseudotime analyses were used to track the origins and changes of meristems throughout the development from primary to secondary vascular tissues.Surprisingly,two types of meristematic-like cell pools within secondary vascular tissues were inferred based on high-resolution microscopy combined with ST,and the results were confirmed by in situ hybridization of,transgenic trees,and single-cell sequencing.The rectangle shape procambium-like(PCL)cells develop from procambium meristematic cells and are located within the phloem domain to produce phloem cells,whereas fusiform shape cambium zone(CZ)meristematic cells develop from fusiform metacambium meristematic cells and are located inside the CZ to produce xylem cells.The gene expression atlas and transcriptional networks spanning the primary transition to secondary vascular tissues generated in this work provide new resources for studying the regulation of meristem activities and the evolution of vascular plants.A web server(https://pgx.zju.edu.cn/stRNAPal/)was also established to facilitate the use of ST RNA-seq data.

A novel protein domain is important for photosystem Ⅱ complex assembly and photoautotrophic growth in angiosperms

Photosystem Ⅱ(PSⅡ)is a multi-subunit protein complex of the photosynthetic electron transport chain that is vital to photosynthesis.Although the structure,composition,and function of PSⅡ have been extensively studied,its biogenesis mechanism remains less understood.Thylakoid rhodanese-like(TROL)provides an anchor for leaf-type ferredoxin:NADP^(+)oxidoreductase.Here,we report the chacterizaton of a second type of TROL protein,TROL2,encoded by seed plant genomes whose function has not previously been reported.We show that TROL2 is a PSⅡ assembly cofactor with essential roles in the establishment of photoautotrophy.TROL2 contains a 45-amino-acid domain,termed the chlorotic lethal seedling(CLS)domain,that is both necessary and sufficient for TROL2 function in PSⅡ assembly and photoautotrophic growth.Phylogenetic analyses suggest that TROL2 may have arisen from ancestral TROL1 via gene duplication before the emergence of seed plants and acquired the CLS domain via evolution of the sequence encoding its N-terminal portion.We further reveal that TROL2(or CLS)forms an assembly cofactor complex with the intrinsic thylakoid membrane protein LOW PSⅡ ACCUMULATION2 and interacts with small PSⅡ subunits to facilitate PSⅡ complex assembly.Collectively,our study not only shows that TROL2(CLS)is essential for photoautotrophy in angiosperms but also reveals its mechanistic role in PSⅡ complex assembly,shedding light on the molecular and evolutionary mechanisms of photosynthetic complex assemblyin angiosperms.

Phosphate-dependent regulation of vacuolar trafficking of OsSPX-MFSs is critical for maintaining intracellular phosphate homeostasis in rice

Vacuolar storage of inorganic phosphate(Pi)is essential for Pi homeostasis in plants.The SPX-MFS family proteins have been demonstrated to be vacuolar Pi transporters in many plant species.Transcriptional regulation of the predominant transporter among rice SPX-MFSs,OsSPX-MFS3,was only moderately suppressed by Pi starvation.Thus,post-transcriptional mechanisms were hypothesized to regulate the activity of OsSPX-MFS3.In this study,we found that the tonoplast localization of OsSPX-MFSs is inhibited under Pi-depleted conditions,resulting in their retention in the pre-vacuolar compartments(PVCs).A yeast two-hybrid screen identified that two SNARE proteins,OsSYP21 and OsSYP22,interact with the MFS domain of OsSPX-MFS3.Further genetic and cytological analyses indicate that OsSYP21 and OsSYP22 facilitate trafficking of OsSPX-MFS3 from PVCs to the tonoplast.Although a homozygous frameshift mutation in OsSYP22 appeared to be lethal,tonoplast localization of OsSPX-MFS3 was significantly inhibited in transgenic plants expressing a negative-dominant form of OsSYP22(OsSYP22-ND),resulting in reduced vacuolar Pi concentrations in OsSYP22-ND plants.Under Pi-depleted conditions,the interaction between OsSYP22 and OsSPX-MFS3 was disrupted,and this process depended on the presence of the SPX domain.Deleting the SPX domains of OsSPX-MFSs resulted in their tonoplast localization under both Pi-depleted and Pi-replete conditions.Complementation of the osspx-mfs1/2/3 triple mutants with the MFS domain or the SPX domain of OsSPX-MFS3 confirmed that the MFS and SPX domains are responsive to Pi transport activity and Pi-dependent regulation,respectively.These data indicated that the SPX domains of OsSPX-MFSs sense cellular Pi(InsP)levels and,under Pi-depleted conditions,inhibit the interaction between OsSPX-MFSs and OsSYP21/22 and subsequent trafficking of OsSPX-MFSs from PVCs to the tonoplast.

Co-regulation of mitochondrial and chloroplast function: Molecular components and mechanisms

The metabolic interdependence,interactions,and coordination of functions between chloroplasts and mitochondria are established and intensively studied.However,less is known about the regulatory components that control these interactions and their responses to external stimuli.Here,we outline how chloroplastic and mitochondrial activities are coordinated via common components involved in signal transduction pathways,gene regulatory events,and post-transcriptional processes.The endoplasmic reticulum emerges as a point of convergence for both transcriptional and post-transcriptional pathways that coordinate chloroplast and mitochondrial functions.Although the identification of molecular components and mechanisms of chloroplast and mitochondrial signaling increasingly suggests common players,this raises the question of how these allow for distinct organelle-specific downstream pathways.Outstanding questions with respect to the regulation of post-transcriptional pathways and the cell and/or tissue specificity of organelle signaling are crucial for understanding how these pathways are integrated at a wholeplant level to optimize plant growth and its response to changing environmental conditions.

Overexpression of OsPAP10a, A Root-Associated Acid Phosphatase, Increased Extracellular Organic Phosphorus Utilization in Rice

Phosphorus （P） deficiency is a major limitation for plant growth and development. Among the wide set of responses to cope with low soil P, plants increase their level of intracellular and secreted acid phosphatases （APases）, which helps to catalyze inorganic phosphate （Pi） hydrolysis from organophosphates, in this study we characterized the rice （Oryza sativa） purple acid phosphatase 10a （OsPAPIOa）. OsPAPIOa belongs to group la of purple acid phosphatases （PAPs）, and clusters with the principal secreted PAPs in a variety of plant species including Arabidopsis. The transcript abundance of OsPAPIOa is specifically induced by Pi deficiency and is controlled by OsPHR2, the central transcription factor controlling Pi homeostasis. In gel activity assays of root and shoot protein extracts, it was revealed that OsPAPIOa is a major acid phosphatase isoform induced by Pi starvation. Constitutive overexpression of OsPAPIOa results in a significant increase of phosphatase activity in both shoot and root protein extracts. In vivo root 5-bromo.4-chloro-3-indolyl-phosphate （BCIP） assays and activity measurements on external media showed that OsPAPIOa is a root-associated APase. Furthermore, overexpression of OsPAPIOa significantly improved ATP hydrolysis and utilization compared with wild type plants. These results indicate that OsPAPIOa can potentially be used for crop breeding to improve the efficiency of P use.

Two soybean bHLH factors regulate response to iron deficiency

Iron is an indispensable micronutrient for plant growth and development. Limited bioavailability of Fe in the soil leads to iron deficiency chlorosis in plants and yield loss. In this study, two soybean basic helix-loop- helix transcription factors, GmbHLH57 and GmbHLH300, were identified in response to Fe-deficiency. Both transcription factors are expressed in roots and nodules, and are induced by Fe deficiency; these patterns were confirmed in transgenic hairy roots expressing constructs of the endogenous promoters fused to a GUS reporter gene. Bimolecular fluorescence complementation, yeast two-hybrid and coimmunoprecipitation （co-IP） assays indicated a physical interaction between GmbHLH57 and GmbHLH300. Studies on transgenic soybeansoverexpressing GmbHLH57 that overexpression of each results in no change of the and GmbHLH300revealed transcription factor, alone, responses to Fe deficiency, whereas overexpression of both transcription factors upregulated the downstream Fe uptake genes and increased the Fe content in these transgenic plants. Compared to wild type, these double overexpression transgenic plants were more tolerant to Fe deficiency. Taken together, our findings establish that GmbHLH57 and GmbHLH300 are important transcription factors involved in Fe homeostasis in soybean.

Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification.In this study,a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin(PVC)sheet.This modified method is named EZ-D,for EASY DNA extraction.Compared with the original cetyl trimethylammonium bromide(CTAB)method,DNA extracted by EZ-D is more efficient in polymerase chain reaction(PCR)amplification due to the more stable performance of the EZ-D stick.The EZ-D method is also faster,easier,and cheaper.PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples.A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL.Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80%in GC content.EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues.Moreover,when EZ-D was combined with the loop-mediated isothermal amplification(LAMP)method,DNA identification of biological samples could be achieved without the need for specialized equipment.As an optimized DNA purification method,EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.