AtCDC5 regulates the G2 to M transition of the cell cycle and is critical for the function of Arabidopsis shoot apical meristem

As a cell cycle regulator, the Myb-related CDC5 protein was reported to be essential for the G2 phase of the cell cycle in yeast and animals, but little is known about its function in plants. Here we report the functional characterization of the CDC5 gene in Arabidopsis thaliana. Arabidopsis CDC5 （AtCDC5） is mainly expressed in tissues with high cell division activity, and is expressed throughout the entire process of embryo formation. The AtCDC5 loss-of-function mutant is embryonic lethal. In order to investigate the function of AtCDC5 in vivo, we generated AtCDC5-RNAi plants in which the expression of AtCDC5 was reduced by RNA interference. We found that the G2 to M （G2/M） phase transition was affected in the AtCDC5-RNAi plants, and that endoreduplication was increased. Additionally, the maintenance of shoot apical meristem （SAM） function was disturbed in the AtCDC5-RNAi plants, in which both the WUSCHEL （WUS）- CLAVATA （CLV） and the SHOOT MERISTEMLESS （STM） pathways were impaired. In situ hybridization analysis showed that the expression of STMwas greatly reduced in the shoot apical cells of the AtCDC5-RNAi plants. Moreover, cyclinB1 or Histone4 was found to be expressed in some of these cells when the transcript of STM was undetectable. These results suggest that AtCDC5 is essential for the G2/M phase transition and may regulate the function of SAM by controlling the expression ofSTMand WUS.

The transcription factor HSFA7b controls thermomemory at the shoot apical meristem by regulating ethylene biosynthesis and signaling in Arabidopsis

The shoot apical meristem(SAM)is responsible for overall shoot growth by generating all aboveground structures.Recent research has revealed that the SAM displays an autonomous heat stress(HS)memory of a previous non-lethal HS event.Considering the importance of the SAM for plant growth,it is essential to determine how its thermomemory is mechanistically controlled.Here,we report that HEAT SHOCK TRAN-SCRIPTION FACTOR A7b(HSFA7b)plays a crucial role in this process in Arabidopsis,as the absence of functional HSFA7b results in the temporal suppression of SAM activity after thermopriming.We found that HSFA7b directly regulates ethylene response at the SAM by binding to the promoter of the key ethylene signaling gene ETHYLENE-INSENSITIVE 3 to establish thermotolerance.Moreover,we demonstrated that HSFA7b regulates the expression of ETHYLENE OVERPRODUCER 1(ETO1)and ETO1-LIKE 1,both of which encode ethylene biosynthesis repressors,thereby ensuring ethylene homeostasis at the SAM.Taken together,these results reveal a crucial and tissue-specic role for HSFA7b in thermomemory at the Arabidopsis SAM.

Control of Rice Embryo Development, Shoot Apical Meristem Maintenance, and Grain Yield by a Novel Cytochrome P450

Angiosperm seeds usually consist of two major parts： the embryo and the endosperm. However, the molec- ular mechanism（s） underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO （GE） gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scuteUum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem （SAM） mainte- nance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78AlO, a GE homolog, also produced bigger seeds, implying a con- served role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance.

Primary carbohydrate metabolism genes participate in heat-stress memory at the shoot apical meristem of Arabidopsis thaliana

In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, little is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana displays an autonomous heat-stress (HS) memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA sequencing, we identified genes participating in establishing the SAM's HS transcriptional memory, including the stem cell (SC) regulators CLAVATA1 (CLV1) and CLV3, HEAT SHOCK PROTEIN 17.6A (HSP17.6A), and the primary carbohydrate metabolism gene FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We demonstrate that sugar availability is essential for survival of plants at high temperature. HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2A) directly regulates the expression of HSP17.6A and FBA6 by binding to the heat-shock elements in their promoters, indicating that HSFA2 is required for transcriptional activation of SAM memory genes. Collectively, these findings indicate that plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.

Emerging Role of the Ubiquitin Proteasome System in the Control of Shoot Apical Meristem Function

The shoot apical meristem （SAM） is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant＇s life. A plethora of different families of transcription factors （TFs） play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system （UPS）. However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors.

The SlTPL3–SlWUS module regulates multi-locule formation in tomato by modulating auxin and gibberellin levels in the shoot apical meristem

Malformed fruits depreciate a plant’s market value.In tomato(Solanum lycopersicum),fruit malformation is associated with the multi-locule trait,which involves genes regulating shoot apical meristem(SAM)development.The expression pattern of TOPLESS3(SITPL3)throughout SAM development prompted us to investigate its functional significance via RNA interference(RNAi)and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9(Cas9)-mediated gene editing.Lower SITPL3 transcript levels resulted in larger fruits with more locules and larger SAMs at the 5 d after germination(DAG5)stage.Differentially expressed genes in the SAM of wild-type(WT)and SITPL3-RNAi plants,identified by transcriptome deep sequencing(RNA-seq),were enriched in the gibberellin(GA)biosynthesis and plant hormone signaling pathways.Moreover,exogenous auxin and paclobutrazol treatments rescued the multi-locule phenotype,indicating that SITPL3 affects SAM size by mediating auxin and GA levels in the SAM.Furthermore,SITPL3 interacted with WUSCHEL(SIWUS),which plays an important role in SAM size maintenance.We conducted RNA-seq and DNA affinity purification followed by sequencing(DAP-seq)analyses to identify the genes regulated by SITPL3 and SIWUS in the SAM and to determine how they regulate SAM size.We detected24 overlapping genes regulated by SITPL3 and SIWUS and harboring an SIWUS-binding motif in their promoters.Furthermore,functional annotation revealed a notable enrichment for functions in auxin transport,auxin signal transduction,and GA biosynthesis.Dual-luciferase assays also revealed that SITPL3 enhances SIWUS-mediated regulation(repression and activation)of SIPIN3 and SIGA2 ox4 transcription,indicating that the SITPL3-SIWUS module regulates SAM size by mediating auxin distribution and GA levels,and perturbations of this module result in enlarged SAM.These results provide novel insights into the molecular mechanism of SAM maintenance and locule formation in tomato and highlight the SITPL3-SIWUS module as a key regulator.

Plant stem cells and their regulations in shoot apical meristems

Stem cells in plants,established during embry-ogenesis,are located in the centers of the shoot apical meristem(SAM)and the root apical meristem(RAM).Stem cells in SAM have a capacity to renew themselves and to produce new organs and tissues indefinitely.Although fully differentiated organs such as leaves do not contain stem cells,cells in such organs do have the capacity to re-establish new stem cells,especially under the induction of phytohormones in vitro.Cytokinin and auxin are critical in creating position signals in the SAM to maintain the stem cell organizing center and to position the new organ primordia,respectively.This review addresses the distinct features of plant stem cells and focuses on how stem cell renewal and differentiation are regulated in SAMs.

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype,microstructure of shoot apical meristem(SAM)and histochemical localization of the GUS gene in comparison with the wild type.Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs(such as smaller leaves,shorter petioles),and slower development and flowering time compared to the wild type.Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM,with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type.In addition,analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant,which suggests that the gene trapped by T-DNA may function in the SAM,and T-DNA insertion could influence the functional activity of the related gene in the mutant,leading to alterations in the SAM and a series of phenotypes in the mutant.

Interplay between the shoot apical meristem and lateral organs

Tissues and organs within a living organism are coordinated,but the underlying mechanisms are not well understood.The shoot apical meristem(SAM)continually produces lateral organs,such as leaves,from its peripheral zone.Because of their close proximity,SAM and lateral organs interact during plant development.Existing lateral organs influence the positions of newly formed organs to determine the phyllotaxis.The SAM not only produces lateral organs,but also influences their morphogenesis.In particular,the SAM promotes leaf polarity determination and leaf blade formation.Furthermore,lateral organs help the SAM to maintain homeostasis by restricting stem cell activity.Recent advances have started to elucidate how SAM and lateral organs patterning and growth are coordinated in the shoot apex.In this review,we discuss recent findings on the interaction between SAM and lateral organs during plant development.In particular,polar auxin transport appears to be a commonly used coordination mechanism.

Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis

The shoot meristem generates the entire shoot system and is precisely maintained throughout the life cycle under various environmental challenges.In this study,we identified a prion-like domain(PrD)in the key shoot meristem regulator SHOOT MERISTEMLESS(STM),which distinguishes STM from other related KNOX1 proteins.We demonstrated that PrD stimulates STM to form nuclear condensates,which are required for maintaining the shoot meristem.STM nuclear condensate formation is stabilized by selected PrD-containing STM-interacting BELL proteins in vitro and in vivo.Moreover,condensation of STM promotes its interaction with the Mediator complex subunit MED8 and thereby enhances its transcriptional activity.Thus,condensate formation emerges as a novel regulatory mechanism of shoot meristem functions.Furthermore,we found that the formation of STM condensates is enhanced upon salt stress,which allows enhanced salt tolerance and increased shoot branching.Our findings highlight that the transcription factor partitioning plays an important role in cell fate determination and might also act as a tunable environmental acclimation mechanism.

Adaptive Cell Segmentation and Tracking for Volumetric Confocal Microscopy Images of a Developing Plant Meristem

Automated segmentation and tracking of cells in actively developing tissues can provide high-throughput and quantitative spatiotemporal measurements of a range of cell behaviors; cell expansion and cell-division kinetics leading to a better understanding of the underlying dynamics of morphogenesis. Here, we have studied the problem of constructing cell lineages in time-lapse volumetric image stacks obtained using Confocal Laser Scanning Microscopy （CLSM）. The novel contribution of the work lies in its ability to segment and track cells in densely packed tissue, the shoot apical meristem （SAM）, through the use of a close-loop, adaptive segmentation, and tracking approach. The tracking output acts as an indicator of the quality of segmentation and, in turn, the segmentation can be improved to obtain better tracking results. We construct an optimization function that minimizes the segmentation error, which is, in turn, estimated from the tracking results. This adaptive approach significantly improves both tracking and segmentation when compared to an open loop framework in which segmentation and tracking modules operate separately.

Molecular Control of Flowering in Response to Day Length in Rice

Flowering at the most appropriate times of the year requires careful monitoring of environmental conditions and correct integration of such information with an endogenous molecular network. Rice （Oryza sativa） is a facultative short day plant, and flowers quickly under short day lengths, as opposed to Arabidopsis thaliana whose flowering is accelerated by longer days. Despite these physiological differences, several genes controlling flowering in response to day length （or photoperiod） are conserved between rice and Arabidopsis, and the molecular mechanisms involved are similar. Inductive day lengths trigger expression of florigenic proteins in leaves that can move to the shoot apical meristem to induce reproductivedevelopment. As compared to Arabidopsis, rice also possesses unique factors that regulate expression of florigenic genes. Here, we discuss recent advances in understanding the molecular mechanisms involved in day length perception, production of florigenic signals, and molecular responses of the shoot apical meristem to florigenic proteins.

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton(Gossypium spp.)is one of the most important fiber crops worldwide.In the last two decades,transgenesis and genome editing have played important roles in cotton improvement.However,genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation.Here,we developed a shoot apical meristem(SAM)cell-mediated transformation system(SAMT)that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton(Gossypium hirsutum),Sea island cotton(Gossypium barbadense),and Asiatic cotton(Gossypium arboreum).Through SAMT,we successfully introduced two foreign genes,GFP and RUBY,into SAM cells of some recalcitrant cotton genotypes.Within 2–3 months,transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants.The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP-and RUBY-positive plants,as well as in their progenies,indicating that the transgenes were stably integrated into the genome and transmitted to the next generation.Furthermore,using SAMT,we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing.In summary,the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.