An overview of pigment gland morphogenesis and its regulatory mechanism

Cotton has enormous economic potential,providing high-quality protein,oil,and fibre.But the comprehensive utilization of cottonseed is limited by the presence of pigment gland and its inclusion.Pigment gland is a common characteristic of Gossypium genus and its relatives,appearing as visible dark opaque dots in most tissues and organs of cotton plants.Secondary metabolites,such as gossypol,synthesized and stored in the cavities of pigment glands act as natural phytoalexins,but are toxic to humans and other monogastric animals.However,only a few cotton genes have been identified as being associated with pigment gland morphogenesis to date,and the developmental processes and regulatory mechanism involved in pigment gland formation remain largely unclear.Here,the research progress on the process of pigment gland morphogenesis and the genetic basis of cotton pigment glands is reviewed,for providing a theoretical basis for cultivating cotton with the ideal pigment gland trait.

Cadmium-Induced Structure Change of Pigment Glands and the Reduction of the Gossypol Content in Cottonseed Kernels

The risk of cotton production on arable land contaminated with heavy metals has increased in recent years.Cotton shows stronger and more extensive resistance to heavy metals,such as cadmium(Cd)than that of other major crops.Here,a potted plant experiment was performed to study Cd-induced alterations in the cottonseed kernel gossypol content and pigment gland structure at maturity in two transgenic cotton cultivars(ZD-90 and SGK3)and an upland cotton standard genotype(TM-1).The results showed that Cd accumulation in cottonseed kernels increased with increasing Cd levels in the soil.The seed kernel Cd content in plants grown on Cd-treated soils was 10-20 times greater than the amount in the corresponding controls.There was a significant difference in Cd accumulation in cottonseed kernels at the 400 and 600μM Cd levels.Cd accumulation was higher in SGK3 and ZD-90 than in TM-1.However,the gossypol content in cottonseed kernels was lower in SGK3 and ZD-90 than in TM-1.There was a negative correlation(r=0.550)between Cd accumulation and the gossypol content in cottonseed kernels.The density of cottonseed kernel pigment glands decreased under Cd stress.This is consistent with the change in gossypol content,which decreased under Cd stress.The damage of the cultivars ZD-90 and SGK3 from Cd poisoning was relatively low under Cd stress,while TM-1 was seriously affected and exhibited Cd sensitivity.Further studies are necessary to understand the cause of the reduced gossypol content in cotton seeds under Cd stress.

Cytochemical localization of H_2O_2 in pigment glands of cotton(Gossypium hirsutum L.)

Programmed cel death （PCD） plays a critical role in the development of plant pigment glands, while H2O2, which is a kind of reactive oxygen species （ROS） produced by the aerobic metabolism of cels, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H2O2 in pigment glands ofGossypium hirsutum L. with 3,3-diaminobenzidine （DAB） staining, 2’,7’-dichlorodihydrolfuorescein diacetate （DCFH2）-DA lfuorescent labeling and CeCl3 cytochemical localization techniques. The results showed that thepigment glandsofG. hirsutum could generate H2O2, and the amount and localization of H2O2 variedat different developmental stages. At the early developmental stage, a smal amount of H2O2 accumulated in the vacuole membrane of pigment gland cels. At the intermediate stage, a large number of H2O2 appeared in the vacuole membrane, while cel wals started to accumulate a smal amount of H2O2. When pigment gland cel degraded, H2O2 mainly accumulated on the chloroplast envelope membrane of inner sheath cels. With the degradation of the sheath cels, H2O2was detected in cel wal and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H2O2 would disappeared. The accumulation sites of H2O2are consistent with the process of PCD of individual gland cels, which started from the degra-dation of intracelular membrane and ended with the degradation of cel wals. Thus H2O2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H2O2 are not associated with the light, and no H2O2 was detected in the secretions of pigment glands.

Single-cell transcriptomic analysis reveals the developmental trajectory and transcriptional regulatory networks of pigment glands in Gossypium bicki

Comprehensive utilization of cottonseeds is limited by the presence of pigment glands and its inclusion gossypol.The ideal cotton has glandless seeds but a glanded plant,a trait found in only a few Australian wild cotton species,including Gossypium bickii.Introgression of this trait into cultivated species has proved to be difficult.Understanding the biological processes toward pigment gland morphogenesis and the associated underlying molecular mechanisms will facilitate breeding of cultivated cotton varieties with the trait of glandless seeds and glanded plant.In this study,single-cell RNA sequencing(scRNA-seg)was performed on 12222 protoplasts isolated from cotyledons of germinating G.bicki seeds 48 h after imbibition.Clustered into 14 distinct clusters unsupervisedly,these cells could be grouped into eight cell populations with the assistance of known cell marker genes.The pigment gland cells were well separated from others and could be separated into pigment gland parenchyma cells,secretory cells,and apoptotic cells.By integrating the pigment gland cell developmental trajectory,transcription factor regulatory networks,and core transcription factor functional validation,we established a model for pigment gland formation.In this model,light and gibberellin were verified to promote the formation of pigment glands.In addition,three novel genes,GbiERF114(ETHYLENE RESPONSE FACTOR 114),GbiZAT11(ZINC FINGER OF ARABIDOPSIS THALIANA 11),and GbiNTL9(NAC TRANSCRIPTION FACTOR-LIKE 9),Were found to affect pigment gland formation.Collectively,these findings provide new insights into pigment gland morphogenesis and lay the cornerstone for future cotton scRNA-seq investigations.

A reference-grade genome assembly for Gossypium bickii and insights into its genome evolution and formation of pigment glands and gossypol

The pigment gland is a morphological characteristic of Gossypium and its related genera.Gossypium bickii(G_(1))is characterized by delayed pigment gland morphogenesis in the cotyledons.In this study,a referencegrade genome of G_(1) was generated,and comparative genomics analysis showed that G_(1) was closest to Gossypium australe(G_(2)),followed by A-and D-genome species.Two large fragment translocations in chromosomes 5 and 13 were detected between the G genome and other Gossypiumgenomes and were unique to the G_(1) and G_(2) genomes.Compared with the G_(2) genome,two large fragment inversions in chromosomes 12 and 13 were detected in G_(1).According to the phylogeny,divergence time,and similarity analysis of nuclear andchloroplast genomes,G_(1)was formedby hybridization between Gossypium sturtianum(C_(1))and a common ancestor of G_(2) and Gossypiumnelsonii(G_(3)).The coordinated expression patterns of pigment gland formation(GoPGF)and gossypol biosynthesis genes in G_(1) were verified to be consistent with its phenotype,and nine genes thatwere related to theprocessof pigmentgland formationwere identified.Anovel gene,GbiCYP76B6,regulated by GoPGF,was found to affect gossypol biosynthesis.These findings offer insights into the origin and evolution of G_(1) and its mechanism of pigment gland formation and gossypol biosynthesis.

Effects of pigment glands and gossypol on somatic cell culture of upland cotton (Gos-sypium hirsutum L.)

The effects of pigment glands and gossypol on the somatic cell culture of upland cotton were studied, using the materials as follows: three pairs of glanded and gland less upland cotton near isogenic lines, TM-1, and Coker 312. The results showed that the pigment glands and gossypol contents in the explants had great inhibiting effect on the induction and growth of callus in somatic cell culture of upland cotton, and the induction rate of callus and the single callus weight of glandless cotton were much higher than those of their glanded near isogenic lines. It was easier to obtain regeneration plants from glandless cotton than from their glandednear isogenic lines. There was a significant inverse correlation between the gossypol contents in the explants and callus induction rate, with the correlation coefficient of -0.84. The vitro gossypol in the medium had some inhibiting effect on the induction and growth of callus, especially for the gland-less cotton. However, a certain concentration of vitro

原发性色素性结节状肾上腺皮质病临床病理分析及文献复习

目的探讨原发性色素性结节状肾上腺皮质病(PPNAD)的临床病理学特征。方法收集首都医科大学附属北京安贞医院2009年1月至2023年6月2例原发性色素性结节状肾上腺皮质病患者的临床资料、影像学检查、病理特征,进行HE染色及免疫组织化学染色,分析其组织形态及免疫组织化学表达特征,并进行相关文献复习。结果例1男,31岁,肿瘤发生在右侧肾上腺。例2女,63岁,肿瘤发生在左侧肾上腺。镜下观察肾上腺皮质均呈多结节状增生,增生的结节主要由较大的颗粒状嗜酸性粒细胞构成,胞质内含不等量脂褐素。免疫表型:Syn、CD56、α-Inhibin、SDHB呈阳性表达,NSE、CK部分呈阳性表达,CgA、S-100、EMA呈阴性表达,Ki-67增殖指数约为2%;病理诊断为原发性色素性结节状肾上腺皮质病。结论原发性色素性结节状肾上腺皮质病较为罕见,诊断时需综合影像学、组织病理学及免疫组织化学染色进行诊断,必要时辅助基因检测以明确诊断。

亚洲棉、比克氏棉和海岛棉种间杂种的合成及性状研究

用（亚洲棉×比克氏棉）Ｆ１双二倍体为母本与海岛棉的不同色素腺体基因型进行杂交，获得（亚洲棉×比克氏棉）×海岛棉种间杂交三种杂种（简称（亚×比）×海三种杂种，下同）．（亚×比）×海三种杂种植株整体性状似海岛棉亲本，但营养生长旺盛，植株高大，并兼有三个亲本的一些形态特性。（亚×比）×海三种杂种花粉高度不育，但雌配子部分可育。海岛棉的不同色素腺体基因型对（亚×比）×海三种杂种的色素腺体在现有较大的影响，其中Ｇｌ２Ｇｌ２Ｇｌ３Ｇｌ３所产生的（亚×比）×海三种杂种，其种子尚有少量色素腺体，但植株为正常色素腺体类型；而ｇｌ２ｇｌ２ｇｌ３ｇｌ３所产生的（亚×比）×海三种杂种虽种子无色素腺体，但植株为少色素腺体类型，Ｇｌｅ２对（亚×比）×海三种杂种的色素腺体的影响与ｇｌ２ｇｌ２ｇｌ３ｇｌ３相同，只是杂种植株色素腺体的减少现象是更明显。此外，本文对比克氏棉子叶色素腺体延缓发生性状的遗传及其转育途径和技术进行了讨论。

抑制性消减杂交方法克隆棉属突变材料腺体发育相关的cDNAs

该文采用抑制性消减杂交方法 (SSH)构建了棉属突变材料“湘棉 18”种子萌发过程中腺体发育形成前后的两个不同时期的SSH差减文库 .通过差示筛选及反式Northern检测 ,获得了色素腺体形成时期特异表达或优势表达的cDNA片段 .结果表明 ,这些cDNA所涉及的基因 ,多数是棉属中新发现的 ,它们与发育调控、细胞凋亡、蛋白质合成等密切相关 .

陆地棉色素腺体与不同棉酚旋光体含量之间的相关性研究

【目的】研究不同基因型棉花不同器官的色素腺体与棉酚旋光体含量之间的相关性。【方法】以6个陆地棉材料中棉所12、中棉所12(无)、珂字棉312、珂字棉312(无)、IF11和冀棉14为对象,精确测定主要器官的色素腺体密度和大小,并采用柱前衍生拆旋方法进行高效液相色谱分析,测定其相应的棉酚旋光体含量,然后对色素腺体和棉酚旋光体含量进行相关性分析。【结果】低酚棉全株无色素腺体,但在各器官中仍能检测到低于0.02%(质量分数)的少量游离棉酚存在。有酚棉各器官均具有典型的色素腺体,其中棉仁的色素腺体最密,苞叶最稀;色素腺体大小(直径)以铃壳最大,花瓣和棉仁最小。不同器官中,总棉酚含量以棉仁最高,苞叶最小;棉仁和花瓣的右旋棉酚含量大于左旋棉酚,而萼片、铃壳、上部叶、中部叶和下部叶的左旋棉酚含量大于右旋棉酚。有酚棉的色素腺体密度与大小之间呈极显著负相关;而总棉酚含量与不同棉酚旋光体含量之间,以及左右旋棉酚含量之间呈极显著正相关。色素腺体密度与总棉酚含量以及不同棉酚旋光体含量之间呈极显著的正相关,而色素腺体大小与右旋棉酚含量之间呈显著的负相关。【结论】棉仁与花瓣的总棉酚含量存在高度的相关性,因此,可以通过花瓣的棉酚含量来判断棉仁的棉酚含量。

棉花不同腺体类型的棉酚高效液相色谱测定及其生物特性表征

运用高效液相(HPLC)方法对新近育成的特殊和普通的无腺体陆地棉及特殊和普通的有腺体陆地棉及海岛棉等多种遗传类型品种材料的棉酚含量进行了比较研究.实验以一反向C18色谱柱为固定相,甲醇 0.5%的醋酸90∶10为流动相,流速为0.8mL·min-1,建立了分离度好、准确而快速的棉酚HPLC测定方法:其最低检测限量3×10-6g/mL-1,精密度(RSD)和准确度(RME)均小于10%,回收率为100.4%～103.2%.分析表明,不同遗传类型品种材料种子的棉酚含量差异极大.通过准确的高效液相分析展示出各种遗传类型尤其是特殊腺体类等珍贵资源.

澳洲野生棉种子叶色素腺体延缓形成的组织结构观察

对５个澳洲野生棉种休眠种子、种子发育过程中的子叶和种子萌发过程中的子叶进行连续切片观察，并以比克氏棉为例进行透视电镜观察。结果表明，具有子叶色素腺体延缓形成性状的澳洲野生棉种及其杂交后代的休眠种子，虽肉眼看不到色素腺体，但具有一群不同于其它细胞的特殊细胞群，其整体结构类似色素腺体的雏形。因此，提出了“色素腺体原”的新概念。澳洲野生棉种在开花后的幼胚器官形态建成期均先出现色素腺体细胞，然后经数次分裂形成一团色素腺体细胞群，挤压周围细胞而形成为色素腺体原，并保持该结构至种子成熟。当种子萌发１６ｈ后，色素腺体细胞解体，形成色素腺体。此外，本研究对澳洲野生棉种子叶色素腺体延缓形成性状的遗传调控等问题进行了讨论。

棉花叶片茸毛和色素腺数量与绿盲蝽抗性的关系

通过网室抗性鉴定和室内观察,研究了不同棉花品种(系)对绿盲蝽的抗性水平及棉花叶片茸毛数量和色素腺密度与绿盲蝽抗性的关系,为抗棉盲蝽棉花品种选育和制定棉盲蝽防治技术策略提供依据。结果表明,不同棉花品种对绿盲蝽的抗性水平存在明显的差异;不同棉花品种(系)叶片茸毛、色素腺点在不同生长期均存在显著差异;棉花叶面、叶背和叶片茸毛总数在苗期与绿盲蝽抗性存在显著负相关,茸毛稀少品种对绿盲蝽的危害具有一定的抗性;叶面叶脉、叶背叶脉及整叶色素腺点数在花铃期与绿盲蝽抗性存在显著正相关。

基于10个棉花腺体相关材料转录组的EST-SSR标记开发

棉花是重要的经济作物,但有毒棉酚的存在,使棉子得不到充分的利用,所以低酚棉育种是棉花育种的重要内容之一。色素腺体是棉酚的储存器官,开发功能型分子标记,加密棉花遗传图谱对色素腺体和其他重要性状相关基因的研究具有重要作用。本试验以10个有腺体和无腺体材料转录组信息为基础开发EST-SSR标记,在12895条1 kb以上的Unigene中共搜索到1546个SSR位点,发生频率和平均距离分别是11.99%和15.99 kb。得到的EST-SSR中,二碱基和三碱基重复是主要的重复类型,分别占30.85%和48.97%,AT/TA和GAA/CTT分别是二碱基和三碱基的优势重复类型。对长度≥20 bp的SSR共设计合成了56对引物,在这10个材料中进行检测,能扩增出清晰条带的有38对,占67.86%,其中呈多态性的有9对,占23.68%,并对其所在的Unigene功能和表达量进行了初步分析。本研究进一步证明了棉花EST-SSR标记开发的可行性,并且为棉花高密度遗传图谱和腺体相关基因的研究奠定了基础。

陆地棉不同色素腺体基因对比克氏棉子叶色素腺体延缓形成性状的遗传效应

用(亚洲棉×比克氏棉)F_1双二倍体作母本与陆地棉的不同色素腺体基因型进行杂交,获得5个具有不同色素腺体基因的[(亚洲棉X比克氏棉)F_1双二倍体X陆地棉]F_1种间三元杂种.不同色素腺体基因对种间三元杂种植株和花器形态性状元明显效应,但对其色素腺体表现和棉酚含量具有较大的影响.用陆地棉单节显性系1(Gl_2Gl_2gl_3gl_3)配制的种间三元杂种完全表现有比克氏棉的子叶色素腺体延缓形成特性;而用双隐性或显性无色素腺体陆地棉配制的种间三元杂种,虽其种子无色素腺体,但植株为少色素腺体类型.用有色素腺体陆地棉和陆地棉单节显性系2(gl_2gl_2Gl_3Gl_3)配制的种间三元杂种,其种子和植株均为有色素腺体类型.因此,用Gl_2Gl_2gl_3gl_3作为陆地棉亲本与比克氏棉及其后代进行杂交和回交,可以提高该性状的选择效果.

陆地棉子叶色素腺体延缓形成性状的表现与组织结构观察

用(亚洲棉×比克氏棉)F1双二倍体作母本与陆地棉单节显性系1(Gl2Gl2gl3gl3)进行杂交和连续回交,育成了具有子叶色素腺体延缓形成特性的陆地棉种质系———ABH0318。该种质的休眠种子观察不到色素腺体,但解剖镜下可看到子叶边缘处有小量稀小的色素腺体。组织切片观察发现,ABH0318休眠种子内含有3种不同类型的色素腺体结构:(1)具有色素腺雏形但无腺腔的色素腺体原,约占总色素腺体或结构的50%左右;(2)发育不全的色素腺体,其体积较小,腺腔内无内含物,约占25%左右;(3)正常色素腺体类型,分布于子叶的边缘,约占25%左右。种子萌发后,第一类色素腺体结构中的细胞迅速溶解,形成腺腔,并迅速形成正常色素腺体,而第二、三类色素腺体结构无明显的变化。

澳洲野生棉种的子叶棉酚动态及与色素腺体形态的关系

对斯特提棉、南岱华棉、澳洲棉、纳尔逊氏棉和比克氏棉五个澳洲野生棉种在种子萌发过程中的子叶棉酚含量进行了测定和分析。结果表明，五个澳洲野生棉种子叶中的棉酚积累过程与无色素腺体陆地棉品种基本相似，其休眠种子和萌发２４ｈ以内的子叶均检测不到棉酚，为典型的低酚棉类型。当种子萌发４８ｈ以后，其子叶开始出现棉酚，但含量极低；在子叶开始出现棉酚１～２ｄ后，棉酚含量开始迅速增加，到种子萌发第９ｄ达到最大值。此外，讨论了澳洲野生棉种的子叶色素腺体延缓形成的组织结构与棉酚积累的关系，以及该性状的遗传调控机理等。

五个澳洲野生棉种色素腺体和棉酚性状研究

本文研究了斯特提棉（Ｇ．ｓｔｕｒｔｉａｎｕｍ）、南岱华棉（Ｇ．ｎａｎｄｅｗａｒｅｎｓｅ）、澳洲棉（Ｇ．ａｕｓ－ｔｒａｌｅ）、纳尔逊氏棉（Ｇ．ｎｅｌｓｏｎｉ）和比克氏棉（Ｇ．ｂｉｃｋｉ）五个澳洲野生棉种的种仁和植株各主要器官的色素腺体和棉酚及其旋光体含量。结果表明，五个棉种均具有种子无色素腺体、植株有色素腺体的特性，但五个棉种植株各器官上色素腺体的分布差异较大，特别是花瓣，其中斯特提棉和南岱华棉的花瓣具有正常的色素腺体，比克氏棉和澳洲棉的花瓣具有少量的色素腺体，纳尔逊氏棉的花瓣无色素腺体。五个棉种种仁棉酚含量极低，但植株各器官均含有较高的棉酚；棉酚旋光体含量测定结果表明，五个棉种的叶片中均只有左旋棉酚，而不含右旋棉酚。

色素腺体和棉酚对陆地棉茎尖培养的影响

本研究以陆地棉三对色素腺体近等基因系为材料 ,研究色素腺体和棉酚对棉花茎尖培养的影响。结果表明 ,外植体的色素腺体和棉酚性状对于棉花茎尖培养有较大的影响 ,有色素腺体棉的茎尖比对应的无色素腺体近等基因系更易诱导产生不定根和再生植株。无色素腺体棉根尖的不定根出生时间晚于对应的有色素腺体近等基因系 7天左右。棉酚试验结果表明 ,培养基中添加外源棉酚对于有色素腺体棉的茎尖培养无显著影响 ,但能促进无色素腺体棉茎尖的培养效果 ,其中加有 0 .1mg/ L外源棉酚的培养基对于无色素腺体棉的茎尖培养效果最佳 ,其不定根和再生植株的诱导率相当于或优于其对应的有色素腺体近等基因系。然而 ,培养基中的棉酚含量过高不利于棉花茎尖培养 ,当培养基中的棉酚含量超过 0 .2 0 mg/ L时 。

陆地棉色素腺体基因的表达

本文利用色素腺密度不同的4个基因型纯合的陆地棉品种组成的6个世代遗传群体,研究了种子等14个器官(或部位)及整株色素腺基因的表达特点。结果表明:器官上色素腺的存在与否可以看作是质量性状,但色素腺在多数器官上的表达不符合寡基因遗传规律,整株色素腺的表现不宜认为是质量性状;器官上的色素腺密度可以认为是下限值为零的具有连续变异的性状,其分布不呈正态,推测与基因间具有较大的互作效应有关。植株不同器官问基因的表达各异,推测与基因表达的特异性或与基因组构成有关。