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 GhREV transcription factor regulate the development of shoot apical meristem in cotton(Gossypium hirsutum)

Background:Manual topping is a routine agronomic practice for balancing the vegetative and reproductive growth of cotton(Gossypium hirsutum)in China,but its cost-effectiveness has decreased over time.Therefore,there is an urgent need to replace manual topping with new approaches,such as biological topping.In this study,we examined the function of Gh REV transcription factors(a classⅢhomeodomain-leucine zipper family,HD-ZIPⅢ)in regulating the development of shoot apical meristem(SAM)in cotton with the purpose of providing candidate genes for biological topping of cotton in the future.Results:We cloned four orthologous genes of At REV in cotton,namely Gh REV1,Gh REV2,Gh REV3,and Gh REV4.All the Gh REVs expressed in roots,stem,leaves,and SAM.Compared with Gh REV1 and Gh REV3,the expression level of Gh REV2 and Gh REV4 was higher in the SAM.However,only Gh REV2 had transcriptional activity.Gh REV2 is localized in the nucleus;and silencing it via virus-induced gene silencing(VIGS)produced an abnormal SAM.Two key genes,Gh WUSA10 and Gh STM,which involved in regulating the development of plant SAM,showed about 50%reduction in their transcripts in VIGS-Gh REV2 plants.Conclusion:Gh REV2 positively regulates the development of cotton SAM by regulating Gh WUSA10 and Gh STM potentially.

Determination of the Photoperiod-Sensitive Inductive Phase in Maize with Leaf Numbers and Morphologies of Stem Apical Meristem

It is vital to determine the effective photoperiods of maize for making full use of tropical germplasm, which is the foundation for determining the effect of latitude and planting date on the development of photoperiod-sensitive maize cultivars. The objective of this study is to determine the photoperiod-sensitive inductive phase using reciprocal transfer between long- day （LD） （15 h d^-1） and short-day conditions （SD） （9 h d^-1）. For Huangzao 4 and CML288, days to tassel and pollen shedding were recorded, and stem apical meristems （SAM） were observed by a laser scanning confocal microscope. The results show that the seedlings are insensitive to photoperiod when they are very young （juvenile）. However, after this period, LD delays flowering and increases the leaf numbers below the inflorescence, and the length of the interval of the photoperiod-sensitive inductive phase is longer under LD conditions than under SD conditions. Transferred from SD to LD, plants show a sudden decrease in leaf numbers once sufficient SD has been received for flower commitment. While transferred from LD to SD, plants have a continuous increase in leaf numbers during the photoperiod sensitive inductive phase under LD conditions. At the same time, when plants are competent to flowers, the obvious morphology is the elongation of maize SAM. There is an obvious variance of the photoperiod sensitive phase under LD and SD conditions in different maize.

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.

不同玉米自交系中导入SAMS基因的转化与检测

以优良玉米自交系丹598、郑58、昌7-2、掖478的茎尖为受体,采用农杆菌介导法将抗旱基因SAMS转入玉米,对转化植株进行PCR检测,共获得阳性植株35株,表明SAMS基因已经整合到玉米基因组中。同时对影响茎尖转化效率的主要因素进行研究。结果表明,侵染时间、菌液浓度、共培养时间和乙酰丁香酮对转化率影响显著。菌液OD600值为0.5～0.7,侵染15min,共培养3d,乙酰丁香酮浓度为100μmol/L时,有利于提高玉米茎尖转化率,其中昌7-2转化率最高。

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.

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.

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.

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.

棉花茎尖分生组织在微粒轰击法基因转化中的应用

利用棉花茎尖分生组织易于再生成完整植株的特点，结合使用基因枪轰击法将ＣＰＴＩ基因导入棉花茎尖分生组织。经过再生、筛选等培养过程已获得若干卡那霉素抗性株。棉花品种、棉株茎尖的发育天数及茎尖附带的下胚轴长度等对转化效果都有不同程度的影响。筛选的起始时间、培养基的转换时间及卡那霉素的浓度梯度等因素对筛选效果有明显影响。

小麦茎尖丛生芽诱导及植株再生

为了筛选不受季节限制的小麦遗传转化受体,研究了3个小麦栽培品种在3种萌发培养基、4种诱导培养基、3种不同诱导时间和3种生根培养基下从茎尖直接诱导丛生芽的最佳条件和方法。结果表明,MB5基本培养基不添加任何激素最适于小麦成熟胚萌发;而在MB5培养基中添加1.0 mg/L TDZ和0.5 mg/L IBA,小麦茎尖丛生芽诱导率最高;单个茎尖形成丛生芽的数目,随诱导时间延长而增加,诱导时间以不超过30 d为宜;品种间的丛生芽诱导率及丛生芽形成数目没有差异;最佳生根培养基为1/2 MS基本培养基添加1.0 mg/LIBA。由丛生芽诱导再生发育形成的植株开花结实正常。这些研究结果为建立基于小麦丛生芽的再生及遗传转化体系提供了方法。

建立甜高粱(Sorghum bicolor)高频、高效再生体系的研究

【目的】建立能源植物甜高粱(Sorghum bicolor L.Moench)的高频、高效离体培养再生体系,为遗传转化奠定基础。【方法】以甜高粱品种凯勒的芽顶端分生组织为外植体,探讨在甜高粱直接诱导丛生芽的培养过程中,无菌实生苗的适合苗龄,适宜的激素组合与比例;并将获得的最佳培养方法应用于其它两个甜高粱品种:M-81E和意达利,以研究此途径的基因型依赖性。【结果】利用3d苗龄的芽顶端分生组织作为外植体容易获得较高的分生组织膨大率。甜高粱丛生芽诱导的适宜激素组合为4.0mg·L-16-苄基腺嘌呤+0.5mg·L-12,4-二氯苯氧乙酸+0.5mg·L-1噻重氮苯基脲,可获得91%的丛生芽诱导频率。适合甜高粱快速生根的激素配方为0.5mg·L-1萘乙酸。通过芽顶端分生组织直接诱导丛生芽途径,每个外植体可诱导出上百个芽,最终产生30—40株再生苗。M-81E和意达利具有与凯勒相似的丛生芽诱导频率及效率。【结论】本研究成功地建立了甜高粱的高频、高效离体培养再生体系,且具有外植体来源不受限制,基因型依赖性弱,遗传稳定性好的优点。

以棉花茎尖分生组织为受体进行基因枪轰击转化的研究

以棉花茎尖分生组织为受体进行基因枪遗传转化,在不受宿主基因型范围和操作时间的限制等方面优于农杆菌介导法和花粉管通道技术。影响棉花茎尖分生组织基因枪遗传转化率的因素较为复杂,轰击前,外植体制备需考虑茎尖分生组织的取材时间、下胚轴保留的长度、外源生长调节剂及活性炭的使用量,以保证其正常生长;轰击后,抗生素筛选的浓度、筛选培养基的转换时间和抗性苗的壮苗也会影响转化效率;此外,DNA和钨粉或金粉的质量、基因枪参数及操作等对转化效果都有一定的作用。除对影响茎尖分生组织基因枪转化率的因素分析以外,还对近年来在棉花茎尖分生组织遗传转化中的最新动态进行了综述,并对目前存在的问题加以讨论。

卧牛锦的组织培养及其种质创新初探

以具有嵌合性状的卧牛锦幼嫩花葶为外植体,进行了组培再生及其种质创新的初步研究。外植体在MS+4.0 mg·L-16-BA+0.3 mg·L-1IBA培养基上诱导形成的愈伤组织,在添加了不同浓度的6-BA和NAA的MS培养基上诱导分化,其中MS+0.01 mg·L-1NAA培养基最适宜卧牛锦愈伤组织的分化及不定芽的形成;再生获得的不定芽有绿色、黄色和嵌合3种类型,其中嵌合类型的诱导率为14.77%,且植株能够正常生长。研究结果表明,通过愈伤途径对卧牛锦的不定芽诱导能够得到新类型的卧牛锦嵌合体,这对于嵌合体植物的种质创新具有重要的应用价值。

植物嵌合体的研究与应用

本文介绍了植物嵌合体的研究现状及其在育种中的应用。对嵌合体的类型、稳定性,在植物组织、器官发育研究中的利用,以及嵌合体在细胞间相互作用和在植物育种中的研究与应用等方面进行了探讨和总结。

水稻幼穗形态发生与顶端分生组织的研究

应用“铸模”扫描电镜法和组织切片技术对水稻幼穗的形态发生过程和顶端分生组织（ Ａｐｉｃａｌｍ ｅｒｉｓｔｅｍ ）进行了系统而细致的研究。研究表明：从营养生长转入到生殖生长早期，水稻生长锥发生了显著的变化，根据苗端分生组织（ Ｓｈｏｏｔ ａｐｉｃａｌｍ ｅｒｉｓｔｅｍ ， Ｓ Ａ Ｍ ）中原基分化的属性，将水稻幼穗早期起源和发育过程分为花序顶端分生组织期（ Ｉｎｆｌｏｒｅｓｃｅｎｃｅ ａｐｉｃａｌ ｍ ｅｒｉｓｔｅｍ ｐｈａｓｅ， Ｉ Ａ Ｍ Ｐ）、小穗顶端分生组织期（ Ｓｐｉｋｅｌｅｔ ａｐｉｃａｌ ｍ ｅｒｉｓｔｅｍ ｐｈａｓｅ， Ｓ Ｐ Ａ Ｍ Ｐ）、花顶端分生组织期（ Ｆｌｏｒａｌ ｍ ｅｒｉｓｔｅｍ ｐｈａｓｅ， Ｆ Ｍ Ｐ）。在这 ３ 个大的发育时期，又根据每一发育时期中的原基分生组织生长发育的程度及先后顺序分别又可分为：花序 ０ 期、花序Ⅰ期、花序Ⅱ期；小穗期Ⅰ期、小穗Ⅱ期、小穗Ⅲ期；内稃原基分化期、浆片原基分化期、雄蕊原基分化期、心皮原基分化期。同时，在研究过程中还发现了一些与前人所不同的形态发生特征，并初步探讨了水稻幼穗早期的起源及分化发育的机理。