In response to insect attack,plants use intricate signaling pathways,including phytohormones,such as jasmonate(JA),ethylene(ET),and salicylic acid(SA),to activate defenses.Maize(Zea mays)is one of the most important staple food crops around the world.Previous studies have shown that the JA and ET signaling play important roles in maize defense against insects,but little is known about whether and how SA regulates maize resistance to insect herbivores.In this study,we ectopically expressed the NahG(salicylate hydroxylase)gene in maize plants(NahG maize)to block the accumulation of SA.It was found that compared with the wild-type(WT)maize,the NahG maize exhibited decreased resistance to the generalist insects Spodoptera litura and Spodoptera frugiperda and the specialist Mythimna separata,and the compromised resistance in the NahG maize was associated with decreased levels of defensive me-tabolites benzoxazinoids(Bxs)and chlorogenic acid(CA).Quantification of simulated S.litura feeding-induced JA,JA-isoleucine conjugate(JA-Ile),and ET in the WT and NahG maize indicated that SA does not regulate JA or JA-Ile,but positively controls ET.We provide evidence suggesting that the SA pathway does not crosstalk with the JA or the ET signaling in regulating the accumulation of Bxs and CA.Tran-scriptome analysis revealed that the bHLH,ERF,and WRKY transcription factors might be involved in SA-regulated defenses.This study uncovers a novel and important phytohormone pathway in maize defense against lepidopterous larvae.

The early development of the endosperm is crucial for balancing the allocation of maternal nutrients to offspring.This process is believed to be evolutionarily associated with genomic imprinting,resulting in parentally biased allelic gene expression.Beyond Fertilization Independent Seed(FIS)genes,the number of imprinted genes involved in early endosperm development and seed size determination remains limited.This study introduces early endosperm-expressed HAIKU(IKU)downstream Candidate F-box 1(ICF1)and ICF2 as maternally expressed imprinted genes(MEGs)in Arabidopsis thaliana.Although these genes are also demethylated by DEMETER(DME)in the central cell,their activation differs from the direct DME-mediated activation seen in classical MEGs such as the FIS genes.Instead,ICF maternal alleles carry pre-established hypomethylation in their promoters,priming them for activation by the WRKY10 tran-scription factor in the endosperm.On the contrary,paternal alleles are predominantly suppressed by CG methylation.Furthermore,we find that ICF genes partially contribute to the small seed size observed in iku mutants.Our discovery reveals a two-step regulatory mechanism that highlights the important role of conventional transcription factors in the activation of imprinted genes,which was previously not fully recognized.Therefore,the mechanism provides a new dimension to understand the transcriptional regu-lation of imprinting in plant reproduction and development.

氮素是影响玉米生长发育、产量和籽粒品质形成所必需的营养元素.为挖掘早期发育的玉米胚乳响应低氮胁迫的关键基因,揭示玉米胚乳抵御低氮胁迫的生理响应及分子机制,本研究在低氮和足氮处理下,对授粉后第6天的自交系B73玉米胚乳进行氨基酸含量、氨基酸衍生物含量分析和转录组测序.生理测定表明,低氮胁迫下,玉米胚乳中10种氨基酸或氨基酸衍生物含量升高,其中苏氨酸、β-氨基异丁酸、组氨酸、β-丙氨酸、赖氨酸含量升高程度最大,其升高范围介于71.1％～153.1％;而其余21种氨基酸或氨基酸衍生物含量降低,其中鸟氨酸、胱氨酸、天冬酰胺、苯丙氨酸、α-氨基丁酸含量下降程度最大,其下降程度范围介于51.6％～65.8％.转录组测序结果表明,与足氮处理相比,低氮胁迫下玉米胚乳中鉴定到3185个显著上调和2612个显著下调的差异表达基因.进一步检测到参与氮代谢途径和氰基氨基酸代谢途径的差异表达基因分别为12和9个,AP2/ERF-ERF、bZIP、WRKY差异表达转录因子家族成员分别为20、10和21个.这些候选基因可能是玉米胚乳抵御低氮胁迫响应的重要基因资源,其为玉米胚乳应答低氮胁迫的分子机制及耐低氮玉米新品种培育奠定基础.

[目的]WRKY是植物特异性转录因子家族之一,参与植物多种生命活动,但在草莓中鲜见WRKY70相关报道.深入解析WRKY70同源基因在草莓响应胁迫过程中的作用,加快分子育种技术应用,培育新草莓种质资源.[方法]用同源克隆法从'红颜'草莓果实中克隆得到FaWRKY70基因,用生物信息学分析其保守结构域、理化性质、蛋白质结构及进化关系等,并结合qRT-PCR数据进行表达模式分析.[结果]FaWRKY70基因全长1 020 bp,编码339个氨基酸;同源基因比对发现,FaWRKY70与苹果、牡丹等同科物种氨基酸序列相似度较高,且同源基因多与植物对生物、非生物胁迫响应相关,暗示FaWRKY70可能参与草莓抵御胁迫的过程;FaWRKY70在草莓不同器官中均有表达,且差异显著,在花中表达量最高,在果实中最低;水杨酸处理后,FaWRKY70基因快速响应,表达量在3 h后达到最高,随后逐渐降低;茉莉酸甲酯处理后FaWRKY70基因受诱导响应程度小,整体呈下调趋势.[结论]FaWRKY70能通过不同响应方式参与草莓生命活动及激素信号转导过程.

植物生长调节剂5-羟色胺(5-HT,5-hydroxy-tryptamine)已应用于农业生产,以提升作物抗旱性,然而其转录水平的分子机制尚不清楚.本研究通过转录组测序、内源激素水平和抗氧化酶活性的综合评价,探讨模拟干旱胁迫下蜡杨梅幼苗响应外源5-羟色胺的生理和分子影响机制.结果表明,50μmol/L的5-HT处理显著提高了蜡杨梅根系中脱落酸和茉莉酸含量,而100 μmol/L的5-HT处理的结果则刚好相反.50 μmol/L的5-HT处理诱导丙二醛含量和超氧化物歧化酶活性显著升高,而过氧化氢含量则显著降低.基于上述两种浓度水平的5-羟色胺处理基因集富集分析结果表明,差异表达基因集主要包括抗氧化酶活性、氧化还原酶活性、生长素和赤霉素介导信号传导、细胞壁生物合成、木聚糖生物合成、果胶代谢、次生代谢物生物合成、苯丙烷和半乳糖醛酸代谢等.与抗氧化酶活性及激素代谢相关的差异表达基因主要为PER、LAC、DHAR和PIN等.通过加权基因共表达网络分析发现8个共表达基因模块与5-羟色胺及干旱胁迫显著相关,其中枢纽基因KAB1218346.1(LOX3)、KAB1219593.1(WRKY53)和KAB1217691.1(CZF1)主要参与激素代谢和转录调控,上述关键基因及其分子调控机制将是今后研究的重要对象.

[目的]WRKY转录因子参与植物对低磷胁迫反应的调控.基于前期苦荞在低磷胁迫下的转录组数据,克隆FtWRKY28基因,预测基因及其编码蛋白的序列结构特征,分析基因在各器官中以及在低磷和激素处理下的表达模式、蛋白的亚细胞定位与转录激活活性,为阐明基因的生物学功能奠定基础.[方法]基于苦荞基因组数据库注释设计特异引物,用逆转录PCR从低磷处理的苦荞根RNA样中克隆FtWRKY28的完整编码序列,用生物信息学方法分析基因与蛋白的结构以及同源蛋白的进化关系,用实时荧光定量分析基因的表达模式,用拟南芥原生质体瞬时表达体系分析蛋白的亚细胞定位,用酵母单杂交分析蛋白的转录激活活性.[结果]FtWRKY28的完整编码序列长876 bp,编码1个含291个氨基酸、有1个WRKY结构域、锌指结构域为C2 H2型的蛋白,归属WRKY家族Ⅱ组.FtWRKY28绝大部分定位于细胞核,具有转录激活活性.FtWRKY28在根中表达水平最高,受低磷、吲哚乙酸、赤霉素3和6-苄氨基嘌呤显著诱导.[结论]FtWRKY28具有转录因子的基本结构与生物化学特征,可能通过生长素、赤霉素、细胞分裂素信号网络的交互作用,调控苦荞在低磷胁迫下的响应过程.

[目的]为了探究草珊瑚[Sarcandra glabra(thunb)nakai]不同器官中内源激素对类胡萝卜素差异积累的可能调控机制.[方法]通过代谢组学方法分析草珊瑚中内源激素和类胡萝卜素代谢物在不同器官间的差异分布情况,结合转录组学技术挖掘草珊瑚类胡萝卜素相关差异酶基因,进一步预测参与调控差异酶基因的转录因子及其激素相关顺式响应元件,从而分析内源激素对类胡萝卜素差异积累的可能调控作用.[结果]吲哚-3-羧酸(indole-3-carboxylic acid)、反式茉莉酸[(-)-jasmonic acid]、二氢茉莉酮酸甲酯(ethyl dihydrojasmonate)、油菜素甾酮(castasterone)、油菜素内酯(brassinolide)等 7 种内源激素在叶中的含量更高,与角黄素(canthaxanthin)、海胆酮(echinenone)、新黄质(neoxanthin)和念珠藻黄素(nostoxanthin)等 8 种差异代谢物等富集部位一致;而脱落酸(abscisic acid,ABA)、赤霉素 4,5-甲氧基水杨酸(gibberellin 4,5-methoxysalicylic acid)、二氢茉莉酸(dihydrojasmonic acid)和 5,6-二羟基吲哚(5,6-dihydroxyindole)在根中的含量更高,与脱落酸醛(abscisic aldehyde)、4,4'-二聚戊烯(4,4'-aiapolycopenedial)、花药黄质(antheraxanthin)这 3 种差异代谢物富集部位一致;关键转录因子MYB106、SPL1、NAC015、ERF064、WRKY44、BHLH116 可通过响应AUX、SA、GA、ABA、Me_JA等植物激素的刺激,参与调控类胡萝卜素合成途径的DXS、VDE、ABA2、AOG、ZEP、CYP97C1、DWARF27、CRTISO、LCYB、PSY这 10 种代谢酶的表达.实时荧光定量PCR(quantitative real-time polymerase chain reaction,RT-qPCR)结果表明,随机选取的 8 个差异基因表达趋势与转录组测序结果一致.[结论]筛选出草珊瑚叶和根与类胡萝卜素相关的 15 种差异内源激素与 11 种差异代谢物,预测了 6 种转录因子参与调控 10 种代谢酶.

[目的]石仙桃Pholidota chinensis Lindl为珍稀濒危的附生兰,附生较地生而言进化出了许多特殊的形态结构和生理生化特性,而转录因子对调控植物的生长发育具有作用,旨在探讨和发掘石仙桃中调控其生长发育的转录因子.[方法]利用PacBio和Illumina测序技术获得石仙桃的全长转录组数据以及在根、根状茎、假鳞茎和叶片中的基因表达量,通过进一步生信分析,探讨石仙桃 4 种转录因子家族成员、理化特性及其组织表达特异性,并用氯化钠(NaCl)和茉莉酸甲酯(MeJA)诱导逆境胁迫,用RT-qPCR分析了C2H2家族和bHLH家族的表达量.[结果]石仙桃中鉴定的4种转录因子及其家族成员转录本为:C2H2家族21个、bHLH家族 27 个、WRKY家族 30 个、bZIP家族 19 个,这 4 个家族及其成员主要为亲水的不稳定性蛋白,亚细胞定位主要位于细胞核内,bHLH家族的部分成员位于细胞质、叶绿体等细胞器中,各基因家族均具有典型的保守结构域特征,在石仙桃的根、根状茎、假鳞茎和叶片呈现出明显的组织特异性.21 个C2H2 家族和 27 个bHLH家族中所有基因表达均受NaCl和MeJA影响,但家族成员受到促进或抑制表达及其表达量存在差异.[结论]鉴定了石仙桃中 4 个转录因子家族成员的数量,各家族成员主要是位于细胞核内的亲水不稳定性蛋白,并具有保守结构域特征和组织表达特异性.

Grain essential amino acid(EAA)levels contribute to rice nutritional quality.However,the molecular mech-anisms underlying EAA accumulation and natural variation in rice grains remain unclear.Here we report the identification of a previously unrecognized auxin influx carrier subfamily gene,OsAUX5,which encodes an amino acid transporter that functions in uptake of multiple amino acids.We identified an elite haplotype of Pro::OsAUX5Hap2 that enhances grain EAA accumulation without an apparent negative effect on agronomic traits.Natural variations of OsAUX5 occur in the cis elements of its promoter,which are differentially acti-vated because of the different binding affinity between OsWRKY78 and the W-box,contributing to grain EAA variation among rice varieties.The two distinct haplotypes were shown to have originated from different Oryza rufipogon progenitors,which contributed to the divergence between japonica and indica.Introduction of the indica-type Pro∷OsAUX5Hap2 genotype into japonica could significantly increase EAA levels,indicating that indica-type Pro∷OsAUX5Hap2 can be utilized to increase grain EAAs of japonica vari-eties.Collectively,our study uncovers an WRKY78-OsAUX5-based regulatory mechanism controlling grain EAA accumulation and provides a potential target for breeding EAA-rich rice.

During effector-triggered immunity(ETI)against the devastating rice blast pathogen Magnaporthe oryzae,Pi9 functions as an intracellular resistance protein sensing the pathogen-secreted effector AvrPi9 in rice.Importantly,the underlying recognition mechanism(s)between Pi9 and AvrPi9 remains elusive.In this study,we identified a rice ubiquitin-like domain-containing protein(UDP),AVRPI9-INTERACTING PROTEIN 1(ANIP1),which is directly targeted by AvrPi9 and also binds to Pi9 in plants.Phenotypic analysis of anip1 mu-tants and plants overexpressing ANIP1 revealed that ANIP1 negatively modulates rice basal defense against M.oryzae.ANIP1 undergoes 26S proteasome-mediated degradation,which can be blocked by both AvrPi9 and Pi9.Moreover,ANIP1 physically associates with the rice WRKY transcription factor OsWRKY62,which also interacts with AvrPi9 and Pi9 in plants.In the absence of Pi9,ANIP1 negatively regulates OsWRKY62 abundance,which can be promoted by AvrPi9.Accordingly,knocking out of OsWRKY62 in a non-Pi9 back-ground decreased immunity against M.oryzae.However,we also observed that OsWRKY62 plays negative roles in defense against a compatible M.oryzae strain in Pi9-harboring rice.Pi9 binds to ANIP1 and OsWRKY62 to form a complex,which may help to keep Pi9 in an inactive state and weaken rice immunity.Furthermore,using competitive binding assays,we showed that AvrPi9 promotes Pi9 dissociation from ANIP1,which could be an important step toward ETI activation.Taken together,our results reveal an immune strategy whereby a UDP-WRKY module,targeted by a fungal effector,modulates rice immunity in distinct ways in the presence or absence of the corresponding resistance protein.