Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana. However, the leaf development that is specific to plants has been little studied. With the aim of contributing to the genetic dissection of leaf development, we have performed a large-scare screening for mutants with abnormal leaves. Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis, four independent mutant lines have been identified and studied genetically. Phenotypes of these mutant lines represent the defects of four novel nuclear genes designated LL1 (LOTUS LEAF 1), LL2 (LOTUS LEAF 2), URO (UPRIGHT ROSETTE), and EIL (ENVIRONT CONDITION INDUCED LESION). The phenotypic analysis indicates that these genes play important roles during leaf development. FOr the further genetic analysis of these genes and the map-based cloning of LL1 and LL2, we have mapped these genes to chromosome regions with an efficient and rapid mapping method.

Effect of Low Light on the Characteristics of Photosynthesis and Chlorophyll a Fluorescence During Leaf Development of Sweet Pepper

Low light stress is one of the main limiting factors which influence the production of sweet pepper under protected cultivation in China. In this experiment, two genotypes of sweet pepper, ShY （low light-tolerant genotype） and 20078 （low light-sensitive genotype）, were used to study the effects of low light （photosynthetic photon flux density, PPFD was 75- 100 umol m-2 s-1, control 450-500 umol m-2 s-1） on photosynthesis during leaf development. The result indicated that under low light chlorophyll content, net photosynthetic rate （PN）, photosynthetic apparent quantum efficiency （Фi） and carboxylation efficiency （CE） of sweet pepper leaves increased gradually and decreased after reaching the maximum levels. The time to reach the peak values for all the above parameters was delayed, whereas the light compensation point （LCP） decreased gradually along with leaf expansion. The decrease in maximum quantum yield of PS II （Fv/Fm） was not observed at any stages of the leaf development under low light condition, but the actual PS II efficiency under irradiance （ФPS II） was lower accompanied by an increased non-photochemical quenching （NPQ） in young and/or old leaves compared with mature leaves. The antenna thermal dissipation （D） was a main way of heat dissipation when young leaves received excessive light energy, while the decline in photosynthetic function in senescence leaf was mostly owing to the decrease in carbon assimilation capacity, followed by a significantly increased allocation of excessive energy （Ex）. Compared with 20078, ShY could maintain higher PN, ФPS II and lower QA reduction state for a longer time during leaf development. Thus, in ShY photosynthetic efficiency and the activity of electron transport of PS II were not significantly affected due to low light stress.

Does overshoot in leaf development of ponderosa pine in wet years leads to bark beetle outbreaks on fine-textured soils in drier years?

Background： Frequent outbreaks of insects and diseases have been recorded in the native forests of western North America during the last few decades, but the distribution of these outbreaks has been far from uniform. In some cases, recent climatic variations may explain some of this spatial variation along with the presence of expansive forests composed of dense, older trees. Forest managers and policy makers would benefit if areas especially prone to disturbance could be recognized so that mitigating actions could be taken. Methods： We use two ponderosa pine-dominated sites in western Montana, U.S.A. to apply a modeling approach that couples information acquired via remote sensing, soil surveys, and local weather stations to assess where bark beetle outbreaks might first occur and why. Although there was a general downward trend in precipitation for both sites over the period between 1998 and 2010 （slope =-1.3, R2 = 0.08）, interannual variability was high. Some years showed large increases followed by sharp decreases. Both sites had similar topography and fire histories, but bark beetle activity occurred earlier （circa 2000 to 2001） and more severely on one site than on the other. The initial canopy density of the two sites was also similar, with leaf area indices ranging between 1.7-2.0 m2. m-2. We wondered if the difference in bark beetle activity was related to soils that were higher in clay content at site I than at site II. To assess this possibility, we applied a process-based stand growth model （3-PG） to analyze the data and evaluate the hypotheses. Results： We found that when wet years were followed by drier years, the simulated annual wood production per unit of leaf area, a measure of tree vigor, dropped below a critical threshold on site I but not on site II. Conclusion： We concluded that the difference in vulnerability of the two stands to beetle outbreaks can be explained largely by differences in gross photosynthesis attributed to the fact that an equivalent amount of stored water in the rooting zone （100 mm） is extracted less efficiently from fine-textured soils than from coarse-textured ones.

The many roles of small RNAs in leaf development

Leaf development involves many complex genetic interactions,signals between adjacent cells or between more distant tissues and consequent changes in cell fate.This review describes three stages in leaf development where regulation by small RNAs have been used to modulate gene expression patterns.

Molecular mechanism of lycopene cyclases regulating carotenoids ratio in different branches during tea leaf and flower development

Carotenoids are essential components in tea quality, contributing to leaf color and aroma. However, little information about carotenoids in different tea cultivars and their biosynthesis regulation mechanism during leaf development is known. Here we analyzed carotenoids by HPLC in the buds and leaves of 113 tea cultivars harvested on the same day. By profile clustering, carotenoids were divided into five groups. Same group cultivars displayed divergence in the total content of carotenoids but a similar molar ratio. To figure out the molecular mechanisms of this phenomenon, we further characterized all functional lycopene cyclases, which are the branch point of the carotenoid biosynthesis pathway. Two β-lycopene cyclases(CsLCYB1 and CsLCYB2) and one ε-lycopene cyclase(CsLCYE1) were cloned. Subcellular localization analysis showed that all cloned CsLCYs were localized in plastids. Enzyme activity assays in E. coli indicated both CsLCYBs catalyzed lycopene into β-carotene, and CsLCYE1 produced δ-carotene and ε-carotene. We found CsLCYB1 and CsLCYE1 predominantly expressed in leaf, while CsLCYB2was mainly expressed during flowering stages. Suppression by antisense oligonucleotides reduced CsLCYB1 and CsLCYE1 transcripts and led to reduction of both β,β-branch and β,ε-branch carotenoids in leaf. The expression levels of CsLCYB1 showed a significant positive correlation withβ,β-branch carotenoids in leaf. Our study provides carotenoid profiles of different tea cultivars, which can assist tea producers in selecting cultivars of interest. Meanwhile, we proposed the molecular mechanism of carotenoids reflecting the tenderness of tea plant leaf from a metabolic flux perspective, and suggested lycopene cyclase that could be applied to the breeding of tea varieties with different branch carotenoids.

Gene expression profiles in early leaf of rice(Oryza sativa)and foxtail millet(Setaria italica)

Plant anatomy is patterned early during leaf development which suggests studying the spatial–temporal transcriptomes of primordia will help identify critical regulative and functional genes.We successfully isolated the leaf primordia tissues from the C3grass rice and the C4grass foxtail millet by laser capture microdissection(LCM)and studied the gene expression throughout leaf developmental stages.Our data analysis uncovered the conserved expression patterns of certain gene clusters both in rice and foxtail millet during leaf development.We revealed genes and transcription factors involved in vein formation,stomatal development,and suberin accumulation.We identified 79 candidate genes associated with functional regulation of C4anatomy formation.Screening phenotype of the candidate genes revealed that knock-out of a putative polar auxin transport related gene NAL1 resulted significantly reduced veinal space in rice leaf.Our present work provides a foundation for future analyses of genes with novel functions in grasses and their role in leaf development,in particular the role in leaves with a contrasting C3vs.C4biosynthetic pathway.

A growth-regulating factor 7(GRF7)-mediated gene regulatory network promotes leaf growth and expansion in sugarcane

Knowledge of the function of growth-regulating factors(GRFs)in sugarcane(Saccharum officinarum and S.spontaneum)growth and development could assist breeders in selecting desirable plant architectures.However,limited information about GRFs is available in Saccharum due to their polyploidy.In this study,22 GRFs were identified in the two species and their conserved domains,gene structures,chromosome location,and synteny were characterized.GRF7 expression varied among tissues and responded to diurnal rhythm.SsGRF7-YFP was localized preferentially in the nucleus and appears to act as a transcriptional cofactor.SsGRF7 positively regulated the size and length of rice leaves,possibly by regulating cell size and plant hormones.Of seven potential transcription factors binding to the SsGRF7 promoter in S.spontaneum,four showed positive expression patterns,and two showed negative expression patterns relative to SsGRF7.

Mutations in the WUSCHEL-related homeobox1 gene cause an increased leaflet number in soybean

WUSCHEL-related homeobox(WOX)transcription factors play a crucial role in lateral organ development in several plant species;however,their precise functions in soybean(Glycine max[L.]Merr.)were unclear.Here,we identified two independent multi-leaflet soybean mutants,mlw48-8 and mlw48-161,from a CRISPR/Cas9-engineered mutant library in the Williams 82 background.Both mutants exhibited irregular leaf margins,and the upper leaves were narrow and almost lanceolate at maturity.Molecular analysis revealed that these are allelic mutants with independent mutations in the WUSCHEL-related homeobox1(GmWOX1A)gene.A transcriptome analysis demonstrated that GmWOX1A modulates the expression of auxin-and leaf development–related genes.Yeast two-hybrid and split-luciferase complementation imaging assays revealed that GmWOX1A interacts with the YABBY family protein GmYAB5,providing further evidence of its potential involvement in leaf development.Notably,the mlw48-161 mutant showed an increased seed number per plant.Consequently,our study not only provides valuable insights into the role of GmWOX1A in soybean leaf development but also offers a potential strategy for high-yield breeding.

Transcriptional,post-transcriptional and post-translational regulations of gene expression during leaf polarity formation

Leaf morphogenesis requires the establishment of adaxlal-abaxlal polarity after primordium initiation from the snoot apical meristem （SAM）. Several families of transcription factors are known to play critical roles in promoting adaxial or abaxial leaf fate. Recently, post-transcriptional gene silencing pathways have been shown to regulate the establishment of leaf polarity, providing novel and exciting insights into leaf development. For example, microRNAs （miR 165/166） and a trans-acting siRNA （TAS3-derived tasiR-ARF） have been shown to repress the expression of several key transcription factor genes. In addition, yet another level of regulation, post-translational regulation, has been revealed recently by studies on the role of the 26S proteasome in leaf polarity. Although our understanding regarding the molecular mecha- nisms underlying establishment of adaxial-abaxial polarity has greatly improved, there is still much that remains elusive. This review aims to discuss recent progress, as well as the remaining questions, regarding the molecular mechanisms underlying leaf polarity formation.

Photosynthetic response of poplar leaves at different developmental phases to environmental factors

With economic incentives and interests in fast-growing poplar trees for short-rotation production of fiber and veneer, many new poplar hybrids have been bred and planted in China, but how to match the new poplar clones to suitable sites and maintain their higher growth rates is still not very clear. In this study, the photosynthetic response of poplar leaves at various developmental stages during two seasons (summer and autumn) was explored and mechanistic models for the photosynthesis of poplar leaves at different developmental phases in response photosynthetic active radiation (PAR), temperature, and relatively humidity were established using the optimization software package 1st Opt. Mature poplar leaves in autumn had significantly higher photosynthetic capacity than leaves at other stages and seasons. Based on the models established for poplar leaves at different phases, the main limiting factors for photosynthesis at the research site were high PAR and temperature in the summer and low PAR in the autumn. Our results highlight the importance of selecting suitable sites, pruning and stand density control during the plantation development to maintain higher photosynthetic rates of poplar trees and to establish optimum cultivation patterns for various utilization of poplar plantations.

OsFK1 encodes C-14 sterol reductase,which is involved in sterol biosynthesis and affects premature aging of leaves in rice

The enzyme C-14 sterol reductase is involved in biosynthesis of brassinosteroids(BR)and sterols,as well as plant development.OsFK1,a member of the sterol biosynthesis pathway located in the endoplasmic reticulum(ER),encodes C-14 sterol reductase.However,there is little research on the function of C-14 sterol reductase in rice.Compared with the wild type,an osfk1 mutant showed dwarf phenotype and premature aging in the second leaf during the trefoil stage,and abnormal development of leaf veins during the tillering stage.The osfk1 mutant showed signs of aberrant PCD,as evidenced by TUNEL staining.This suggested that high ROS buildup caused DNA damage and ROS-mediated cell death in the mutant.The osfk1 mutant also showed decreased chlorophyll content and aberrant chloroplast structure.Sequencing of the osfk1 mutant allele revealed a non-synonymous G to A mutation in the final intron,leading to early termination.Here,we identified the OsFK1 allele,cloned it by Mutmap sequencing,and verified it by complementation.HPLC-MS/MS assays demonstrated that the osfk1 mutation caused lower phytosterol levels.These findings showed that the OsFK1 allele encoding C-14 sterol reductase is involved in phytosterol biosynthesis and mediates normal development of rice plants.

不同温度下杉木凋落叶分解过程中碳氮磷释放及其化学计量比变化

[目的]研究升温对不同发育阶段杉木凋落叶分解过程中碳(C)、氮(N)、磷(P)释放规律及其生态化学计量特征的影响,揭示气候变暖背景下杉木人工林凋落叶分解过程中养分释放特征。[方法]收集中龄林(18年生)、成熟林(30年生)和过熟林(42年生)3个发育阶段的杉木凋落叶,设置25、30和35℃3个温度梯度进行室内模拟分解试验。[结果](1)在264 d的分解周期内,各发育阶段杉木凋落叶C、N、P残留率总体表现为随分解时间的增加而减小,但不同元素残留率变化模式不同,C残留率表现为释放—富集—释放模式,N残留率表现为富集—释放模式,P残留率表现为释放—富集模式。(2)拟合模型结果表明,成熟林与过熟林凋落叶分解过程中N周转期在35℃处理下比25℃处理分别缩短了34.4%和16.9%,P周转期分别缩短了38.4%和43.8%。(3)不同发育阶段杉木凋落叶分解过程中C:N、N:P比总体呈波动变化,C:P比呈先增大后减小的变化趋势,杉木凋落叶C:N、C:P、N:P比变幅分别为9.32~39.0、949~2194、32.7~153,升温处理在凋落叶分解过程总体上增大了C:P、N:P,降低了C:N比。[结论]温度升高能够缩短成熟林、过熟林凋落叶分解过程中N、P的周转期,提高各发育阶段杉木凋落叶分解过程中的C:P、N:P,说明杉木凋落叶分解明显受P限制,建议根据不同发育阶段杉木的生长需求,适当增加磷肥的施用。

陆地棉叶片干质量的发育遗传研究

采用基因型与环境互作的加性-显性-母体遗传的非条件与条件分析即ADM遗传模型,研究7个陆地棉亲本及21个正交F1代在3种密度(2.4×10^(5)株/hm^(2)、1.8×10^(5)株/hm^(2)、1.2×10^(5)株/hm^(2))种植下,叶片干质量(LDW)发育遗传规律,探讨陆地棉叶片不同发育时期干质量、发育特征值与发育时间的关系,为优质亲本选育提供依据。结果表明:显性及其与环境互作效应存在于整个叶片干质量累积过程,在叶片不同发育时期或时段也存在较大的加性或母体效应影响,加性及母体与环境互作总体来说影响较小,叶片发育过程中以遗传主效应为主,而遗传主效应表达受环境调控较大;叶片干质量表现出明显杂种优势且易受环境条件影响的特点。不同发育时期或时段叶片干质量对最终叶片干物质的累积均有较大正向作用,但其遗传机制却各不相同。第20天、第30天对叶干质量具有较好选择效果。叶片干物质累积与叶片生长发育特点及亲本的选择有关,可根据各亲本的遗传效应预测值、环境稳定性进行陆地棉叶型育种。7个亲本中,大叶型育种时选择P5为亲本,小叶型育种时选择P2为亲本,以保证营养物质的有效储存与运输。

当归茎、叶发育解剖学研究

以当归幼苗及 2年生植株为材料 ,对其茎、叶的解剖结构进行了观察 ,主要结果为 :①当归无论幼茎或老茎或老茎横切面轮廓均不规则 ,维管束粗细相差较大 ;②紫茎当归和绿茎当归茎叶结构基本一致 ,而造成当归茎颜色差异的原因是遗传基因还是环境条件的影响 ,有待于进一步研究 ;③叶片角质层不发达 ,叶肉内栅栏组织只有 1层 ,海棉组织中有大的细胞间隙 ,由此说明当归属喜阴湿、不耐干旱的植物类型 .

北柴胡茎、叶的发育解剖学研究

目的研究北柴胡茎、叶的个体发育过程。方法应用常规石蜡切片方法对北柴胡(Bupleu-rum chinense DC.)的茎尖、1年生和2年生茎以及叶进行了横向与纵向解剖观察。结果北柴胡茎的发育包括原分生组织、初生分生组织、初生结构和次生生长4个阶段。原分生组织由原套和原体组成,其衍生细胞形成初生分生组织。初生分生组织由原表皮、基本分生组织和原形成层组成。初生分生组织的衍生细胞分化形成茎的初生结构,包括表皮、皮层、维管束、髓和髓射线。随着茎的继续发育,维管形成层开始活动,由束中形成层产生次生韧皮部和次生木质部分子,而束间形成层仅产生薄壁细胞形成宽的射线。茎始终未产生周皮,而叶的发育包括原分生组织、初生分生组织和成熟结构3个阶段。北柴胡的叶为典型的异面叶,由叶片和叶柄两部分组成。分泌道在茎中主要分布在皮层和髓中,在叶中分布在维管束的上、下组织中,且这些分泌道均属初生分泌道。结论北柴胡茎、叶的个体发育过程,类似多年生草本双子叶植物茎、叶的一般发育过程。

东灵山主要树种在不同环境梯度下的叶功能性状研究

以不同发育阶段的辽东栎和五角枫为对象,研究了其5种叶功能性状在不同海拔和不同坡位上的变化。结果表明:1)2个树种的比叶面积和叶干物质含量在不同坡位上都表现出显著的差异,比叶面积为下坡位>上坡位,叶干物质含量与之相反,且不同发育阶段的比叶面积和叶干物质含量随坡位变化的规律性相同,表明不同发育阶段个体的比叶面积和叶干物质含量对坡位变化的响应相同。叶的氮、磷含量和氮磷比在不同坡位间没有显著差异,表明坡位对叶的氮、磷元素影响不大。2)树种不同发育阶段的比叶面积、叶干物质含量、叶氮含量和叶磷含量随着海拔梯度的变化都有显著差异,且变化规律不同;表明不同树种各发育阶段的叶功能性状对海拔梯度变化的响应机制不同。氮磷比在不同海拔高度没有显著差异,可能是因为在较小的生境范围内,海拔对其影响较小。

香樟凋落叶分解物对辣椒生长发育的影响

采用盆栽试验,研究了香樟（Cinnamomum cam phora）凋落叶（0、25、50、75、100 g凋落叶分别与10 kg土壤混合）分解过程中对辣椒（Capsicum annuum）及其子代生长发育的影响,并采用气质联用（GC MS）技术对凋落叶分解前后的萜类物质进行了检测和对比.结果显示：（1）香樟凋落叶分解物显著地抑制了辣椒的叶片数量、整株叶面积以及株高和基径生长,抑制效应随凋落叶剂量的增大而增强,而分解过的凋落叶和塑料片替代凋落叶原样的试验并不抑制辣椒的生长.（2）观察期内（处理45～76 d）,辣椒的现蕾数、开花数和结实数在各凋落叶处理下均明显低于同期对照;采用指数方程拟合的现蕾、开花和结实动态显示,25 g凋落叶处理使辣椒的始蕾期、始花期和始果期分别推迟了0.81、0.17和1.35d,50 g处理使这3项参数分别推迟了4.69、5.78和6.27d,而75 g和100 g处理均使这3项参数推迟10d以上.（3）辣椒的单株果实产量在凋落叶处理下降低29.44％～61.29％,而单果重受到的影响相对较小.（4）辣椒产出的种子千粒重和子代的生长状况并未受到凋落叶分解物的影响.（5）凋落叶的正己烷提取物中萜类占78.05％,而分解后其相对总量下降至40.76％,峰面积下降至原样的约1％.研究认为：香樟凋落叶添加处理对辣椒营养生长和生殖生长产生的明显抑制作用,不是由凋落叶添加对土壤通气透水性或对受体生长造成机械阻隔引起的,其在分解过程中释放樟脑（Camphor）、1,8-桉叶油醇（1,8-cineole）等萜类物质引起化感作用可能才是关键原因.

荷花玉兰叶生长发育的解剖学研究

为探明荷花玉兰叶各发育阶段的组织结构特点及其分泌腔的发育特征,为荷花玉兰叶分泌结构与化学成分的研究和开发利用提供解剖学资料,采用石蜡切片结合显微观察的方法,对荷花玉兰叶发育的各个阶段进行了解剖学研究。结果表明:荷花玉兰叶的生长发育阶段可分为分生组织、幼叶和成熟叶等3个阶段。各阶段生理结构特点分别表现为:分生组织阶段具有分泌腔;幼叶下表皮具大量单细胞表皮毛,分泌腔消失;成熟叶无表皮毛,叶肉分化为栅栏组织和海绵组织。

不同生态区烤烟叶片发育超微结构的对比研究

利用超微技术对云南和河南烤烟叶片的细胞超微结构进行了比较研究。结果表明:在烟叶发育过程中,两地烟叶的超微结构变化规律基本一致,主要差异表现在淀粉粒与嗜锇颗粒上:在叶片发育早期,云南烟叶细胞中叶绿体及淀粉粒明显少于河南烟叶;在叶片发育中期,云南烟叶淀粉粒较多,嗜锇颗粒较少;在叶片发育后期,河南烟叶叶绿体降解迅速,嗜锇颗粒较多。

高品质陆地棉铃-叶系统干物质质量的发育遗传研究

应用加性-显性发育遗传模型及条件与非条件的统计分析方法,对6个高品质陆地棉品种(系)及其30个F1组合的伏桃与对位果枝叶的干物质质量进行了研究。非条件方差分析表明,果枝叶质量在棉铃体积增大期主要受加性效应控制,内部充实期主要受显性效应控制;铃壳质量在不同发育时期均以加性效应为主;铃重在前中期主要受显性效应控制,后期以加性效应为主。条件遗传分析表明,影响果枝叶质量的净遗传效应在铃龄38 d至45 d达到高峰;控制铃壳质量的基因表达分别在铃龄17 d至24 d和铃龄38 d至45 d出现两个高峰;影响铃重性状的基因分别在铃龄17 d至24 d和铃龄31 d至38 d出现两个表达活跃的高峰,此后基因的表达量急剧下降。遗传相关分析表明不同发育时期的铃壳质量均与最终铃重成极显著的加性正相关。