转录因子ABI4调控草莓果实成熟的分子机制

[目的]探讨转录因子ABI4在草莓果实发育过程中的作用。[方法]以八倍体草莓品种‘甜查理’为试材,利用基因同源克隆技术获得了1个草莓转录因子Fa ABI4编码基因,运用Clustal X软件和MEGA 5软件分析了草莓中Fa ABI4与其他物种间的序列同源性和亲缘关系,并利用荧光定量PCR技术分析了Fa ABI4基因随着草莓果实发育的表达模式。通过植物双元表达载体p BI121构建的基因瞬时表达系统对草莓果实中的Fa ABI4基因表达水平进行了调控,Fa ABI4基因表达量瞬时调控出现表型后,分别采取空载体p BI121和干扰载体p BI121-Fa ABI4侵染过的草莓果实并且去掉种子,分析其果实色素含量、脱落酸含量、果实硬度和蔗糖含量等生理指标,以及Fa CHS和Fa UFGT基因的表达水平变化。[结果]获得了1个编码330个氨基酸的Fa ABI4转录因子,与AP2家族的特征一样,含有1个57个氨基酸残基的AP2结构区域,与其他物种如梅花、葡萄和苹果的亲缘关系很近。Fa ABI4基因表达量在草莓果实发育前期很低,在果实发育中期和后期表达量剧烈上升。草莓果实瞬时干扰Fa ABI4基因14 d后发现,与注射空载体p BI121相比,注射p BI121-Fa ABI4的草莓果实表面出现了部分不能正常上色现象。切取不能上色部分的果肉进行生理和分子指标分析发现,与对照果实p BI121相比,p BI121-Fa ABI4降低了果实花色苷和蔗糖含量,促使果实硬度变大,但对ABA含量未有影响,同时降低了花色苷合成路径中的2个关键酶基因Fa CHS和Fa UFGT的表达水平。[结论]草莓中的转录因子Fa ABI4跟其他物种一样具有典型的AP2结构域,属于AP2家族。Fa ABI4正向调控草莓果实发育,可通过影响Fa CHS和Fa UFGT基因表达水平影响花色苷积累,同时可影响果实硬度及蔗糖积累,最终调控草莓果实的发育成熟进程。

ABI4调控侧根发育研究(综述)

生长素是调控植物侧根发育的关键植物激素,生长素运输载体PIN蛋白介导其极性分布。ABI4抑制生长素极性运输蛋白基因PIN1的表达,影响生长素的极性运输,抑制侧根形成。本文概述ABI4转录因子调控侧根发育的研究进展。

PIF4 interacts with ABI4 to serve as a transcriptional activator complex to promote seed dormancy by enhancing ABA biosynthesis and signaling

Transcriptional regulation plays a key role in the control of seed dormancy,and many transcription factors(TFs)have been documented.However,the mechanisms underlying the interactions between different TFs within a transcriptional complex regulating seed dormancy remain largely unknown.Here,we showed that TF PHYTOCHROME-INTERACTING FACTOR4(PIF4)physically interacted with the abscisic acid(ABA)signaling responsive TF ABSCISIC ACID INSENSITIVE4(ABI4)to act as a transcriptional complex to promote ABA biosynthesis and signaling,finally deepening primary seed dormancy.Both pif4 and abi4 single mutants exhibited a decreased primary seed dormancy phenotype,with a synergistic effect in the pif4/abi4 double mutant.PIF4 binds to ABI4 to form a heterodimer,and ABI4 stabilizes PIF4 at the protein level,whereas PIF4 does not affect the protein stabilization of ABI4.Subsequently,both TFs independently and synergistically promoted the expression of ABI4 and NCED6,a key gene for ABA anabolism.The genetic evidence is also consistent with the phenotypic,physiological and biochemical analysis results.Altogether,this study revealed a transcriptional regulatory cascade in which the PIF4–ABI4 transcriptional activator complex synergistically enhanced seed dormancy by facilitating ABA biosynthesis and signaling.

Regulation of cryptochrome-mediated blue light signaling by the ABI4–PIF4 module

ABSCISIC ACID-INSENSITIVE 4(ABI4) is a pivotal transcription factor which coordinates multiple aspects of plant growth and development as well as plant responses to environmental stresses.ABI4has been shown to be involved in regulating seedling photomorphogenesis;however,the underlying mechanism remains elusive.Here,we show that the role of ABI4 in regulating photomorphogenesis is generally regulated by sucrose,but ABI4 promotes hypocotyl elongation of Arabidopsis seedlings under blue(B) light under all tested sucrose concentrations.We further show that ABI4 physically interacts with PHYTOCHROME INTERACTING FACTOR 4(PIF4),a well-characterized growth-promoting transcription factor,and post-translationally promotes PIF4 protein accumulation under B light.Further analyses indicate that ABI4 directly interacts with the B light photoreceptors cryptochromes(CRYs) and inhibits the interactions between CRYs and PIF4,thus relieving CRY-mediated repression of PIF4 protein accumulation.In addition,while ABI4 could directly activate its own expression,CRYs enhance,whereas PIF4 inhibits,ABI4-mediated activation of the ABI4 promoter.Together,our study demonstrates that the ABI4–PIF4 module plays an important role in mediating CRY-induced B light signaling in Arabidopsis.

植物ABI4转录因子的研究进展

Abscisic acid-insensitive 4(ABI4)属于APETALA2/ethy lene responsive factor(AP2/ERF)类转录因子,它是在研究ABA信号转导途径中被发现和鉴定的。近年来,ABI4被认为是一种具有多种调节功能的转录因子,它参与了植物种子萌发和幼苗形态建成、质体/线粒体反向信号传递、脂类合成和分解、糖信号和ABA应答等诸多重要的生物学事件。本文综述了ABI4转录因子的结构特征、转录调控模式、基因表达调控和参与的信号转导功能等的最新研究进展。

Abscisic Acid Antagonizes Ethylene Production through the ABI4-Mediated Transcriptional Repression of ACS4 and ACS8 in Arabidopsis

Increasing evidence has revealed that abscisic acid （ABA） negatively modulates ethylene biosynthesis, although the underlying mechanism remains unclear. To identify the factors involved, we conducted a screen for ABA-insensitive mutants with altered ethylene production in Arabidopsis. A dominant allele of ABI4, abi4-152, which produces a putative protein with a 16-amino-acid truncation at the C-terminus of ABI4, reduces ethylene production. By contrast, two recessive knockout alleles of ABI4, abi4-102 and abi4-103, result in increased ethylene evolution, indicating that ABI4 negatively regulates ethylene produc- tion. Further analyses showed that expression of the ethylene biosynthesis genes ACS4, ACSS, and AC02 was significantly decreased in abi4-152 but increased in the knockout mutants, with partial dependence on ABA. Chromatin immunoprecipitation-quantitative PCR assays showed that ABI4 directly binds the pro- moters of these ethylene biosynthesis genes and that ABA enhances this interaction. A fusion protein containing the truncated ABI4-152 peptide accumulated to higher levels than its full-length counterpart in transgenic plants, suggesting that ABI4 is destabilized by its C terminus. Therefore, our results demon- strate that ABA negatively regulates ethylene production through ABI4-mediated transcriptional repression of the ethylene biosynthesis genes ACS4 and ACS8 in Arabidopsis.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis

Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1(DES1)and the production of H2S in guard cells.However,it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals.In this study,we discovered that ABSCISIC ACID INSENSITIVE 4(ABI4)acts downstream of DES1 in the control of ABA responses in Arabidopsis.ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA,and loss of DES1 function impairs this process.Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination,seedling establishment,and stomatal closure,which are abolished in the ABI4Cys250Ala mutated variant.Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA.Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18(MAPKKK18),which propagates the MAPK signaling cascade induced by ABA.Furthermore,the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18,and ABI4 can bind the DES1 promoter,forming a regulatory loop.Taken together,these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

The DELLA-ABI4-HY5 module integrates light and gibberellin signals to regulate hypocotyl elongation

Plant growth is coordinately controlled by various environmental and hormonal signals,of which light and gibberellin(GA)signals are two critical factors with opposite effects on hypocotyl elongation.Although interactions between the light and GA signaling pathways have been studied extensively,the detailed regulatory mechanism of their direct crosstalk in hypocotyl elongation remains to be fully clarified.Previously,we reported that ABA INSENSITIVE 4(ABI4)controls hypocotyl elongation through its regulation of cellelongation-related genes,but whether it is also involved in GA signaling to promote hypocotyl elongation is unknown.In this study,we showthat promotion of hypocotyl elongation by GA is dependent on ABI4 activation.DELLAs interact directly with ABI4 and inhibit its DNA-binding activity.In turn,ABI4 combined with ELONGATED HYPOCOTYL 5(HY5),a key positive factor in light signaling,feedback regulates the expression of the GA2ox GA catabolism genes and thus modulates GA levels.Taken together,our results suggest that the DELLA-ABI4-HY5 module may serve as a molecular link that integrates GA and light signals to control hypocotyl elongation.

十字花科植物ABI4序列演化与转录激活活性分析

转录因子ABI4参与植物激素ABA的绝大多数生物学功能,它不仅是ABA和GA在植物体内含量平衡的核心调控因子,同时还连接ABA与植物细胞内多个信号通路。利用拟南芥(Arabidopsisthaliana)ABI4序列在十字花科其它19种植物(除拟南芥外)中检索得到27个同源基因,通过序列多态性分析、系统发生重建、染色体共线性分析和转录激活活性比较,探讨了该基因在十字花科植物中的演化历史。结果表明,ABI4蛋白质序列和结构在十字花科植物中具有较高的保守性,暗示了其功能的重要性;单独的ABI4蛋白并不具备显著的转录激活活性,说明其生物学功能的具体分子机制可能相对复杂,需要进一步探讨。

利用基因组重测序技术鉴定一个拟南芥ABA不敏感突变体

脱落酸(ABA)是调控种子休眠与萌发过程的重要植物激素之一。本研究通过筛选拟南芥(Arabidopsis thaliana)T-DNA插入突变体库,获得一株ABA不敏感突变体abi10(ABA-insensitive mutant 10)。遗传分析表明,该突变体受核编码的隐性单基因控制。对abi10突变体植株进行基因组重测序,通过序列比对分析发现abi10突变体基因组中有3个T-DNA插入位点,其中有一个插入位点位于编码ABA信号转导途径中重要转录因子ABI4的基因启动子区。通过与abi4突变体做杂交进行等位突变验证,发现杂交F1代表现为ABA不敏感的隐性表型。进一步通过实时荧光定量PCR检测,发现abi10突变体中ABI4基因的表达量显著降低。综上所述,本文利用基因组重测序技术,鉴定到的abi10是一个新的ABI4基因等位突变体,研究结果为后续深入研究ABI4基因的表达调控机制提供研究材料。

质体反向信号研究进展

细胞内由叶绿体向细胞核发送的信号被称作质体反向信号。它可以协调细胞核与叶绿体编码的基因表达,促进叶绿体发育,并且参与调控植物多种生长发育过程。近年来已经鉴定出一些参与质体反向过程的信号及其转导的组分,为研究质体反向信号的调控网络提供了新视角。本文回顾了质体反向信号的研究历史,并对质体反向信号转导途径及其参与调节生长发育进程等方面的最新进展进行概述。

A Dual-Function Transcription Factor, AtYY1, Is a Novel Negative Regulator of the Arabidopsis ABA Response Network

Abscisic acid （ABA） plays crucial roles in plant growth and development, as well as in response to various environmental stresses. To date, many regulatory genes involved in the ABA response network have been identified; however, their roles have remained to be fully elucidated. In this study, we iden- tified AtYY1, an Arabidopsis homolog of the mammalian C2H2 zinc-finger transcription factor Yin Yang 1 （YY1）, as a novel negative regulator of the ABA response. AtYY1 is a dual-function transcription factor with both repression and activation domains. The expression of AtYY1 was induced by ABA and stress conditions including high salt and dehydration. The yyl mutant was more sensitive to ABA and NaCI than the wild-type, while overexpressing AtYY1 plants were less sensitive. AtYY1 loss also enhanced ABA-induced stomatal closing and drought resistance. Moreover, AtYYI can bind the ABA REPRESSOR1 （ABR1） promoter and directly upregulate ABR1 expression, as well as negatively regulate ABA- and saR-responsive gene expression. Additional analysis indicated that ABA INSENSITIVE4 （ABI4） might positively regulate AtYY1 expression and that ABR1 can antagonize this regulation. Our findings provide direct evidence that AtYY1 is a novel negative regulator of the ABA response network and that the ABI4-AtYY1-ABR1 regulatory pathway may fine-tune ABA-responsive gene expression in Arabidopsis.

Characterization of Evolutionarily ConservedMotifs Involved in Activity and Regulation of theABA-INSENSITIVE （ABI） 4 Transcription Factor

In recent years, the transcription factor ABI4 has emerged as an important node of integration for externaland internal signals such as nutrient status and hormone signaling that modulates critical transitions during the growthand development of plants. For this reason, understanding the mechanism of action and regulation of this protein rep-resents an important step towards the elucidation of crosstalk mechanisms in plants. However, this understanding hasbeen hindered due to the negligible levels of this protein as a result of multiple posttranscriptional regulations. To betterunderstand the function and regulation of the ABI4 protein in this work, we performed a functional analysis of severalevolutionarily conserved motifs. Based on these conserved motifs, we identified ortholog genes of ABI4 in differentplant species. The functionality of the putative ortholog from Theobroma cacao was demonstrated in transient expres-sion assays and in complementation studies in plants. The function of the highly conserved motifs was analyzed aftertheir deletion or mutagenesis in the Arabidopsis ABI4 sequence using mesophyll protoplasts. This approach permitted usto immunologically detect the ABI4 protein and identify some of the mechanisms involved in its regulation. We identi-fied sequences required for the nuclear localization （AP2-associated motif） as well as those for transcriptional activationfunction （LRP motif）. Moreover, this approach showed that the protein stability of this transcription factor is controlledthrough protein degradation and subcellular localization and involves the AP2-associated and the PEST motifs. We dem-onstrated that the degradation of ABI4 protein through the PEST motif is mediated by the 26S proteasome in responseto changes in the sugar levels.

Multifaceted Signaling Networks Mediated by Abscisic Acid Insensitive 4

Although ABSCISIC ACID INSENSITIVE 4(ABI4)was initially demonstrated as a key positive regulator in the phytohormone abscisic acid(ABA)signaling cascade,multiple studies have now shown that it is actually involved in the regulation of several other cascades,including diverse phytohormone biogenesis and signaling pathways,various developmental processes(such as seed dormancy and germination,seedling establishment,and root development),disease resistance and lipid metabolism.Consistent with its versatile biological functions,ABI4 either activates or represses transcription of its target genes.The upstream regulators of ABI4 at both the transcription and post-transcription levels have also been documented in recent years.Consequently,a complicated network consisting of the direct target genes and upstreamregulators of ABI4,through which ABI4 participates in several phytohormone crosstalk networks,has been generated.In this review,we summarize current understanding of the sophisticated ABI4-mediated molecular networks,mainly focusing on diverse phytohormone(including ABA,gibberellin,cytokinin,ethylene,auxin,and jasmonic acid)crosstalks.We also discuss the potential mechanisms through which ABI4 receives the ABA signal,focusing on protein phosphorylation modification events.

A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions

Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely unexplored.Here,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis thaliana.ABI4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR emergence.Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4.MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence.We propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.