a-seed-coat-bedding-assay-to-genetically-explore-vitro-how-endosperm

A Seed Coat Bedding Assay to Genetically Explore <em>In Vitro</em> How the Endosperm Controls Seed Germination in <em>Arabidopsis thaliana</em>

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Université de Genève

Mammalian circadian rhythms are generated by a feedback loop in which BMAL1 and CLOCK, players of the positive limb, activate transcription of the cryptochrome and period genes, components of the negative limb. Bmal1 and Per transcription cycles display nearly opposite phases and are thus governed by different mechanisms. Here, we identify the orphan nuclear receptor REV-ERBalpha as the major regulator of cyclic Bmal1 transcription. Circadian Rev-erbalpha expression is controlled by components of the general feedback loop. Thus, REV-ERBalpha constitutes a molecular link through which components of the negative limb drive antiphasic expression of components of the positive limb. While REV-ERBalpha influences the period length and affects the phase-shifting properties of the clock, it is not required for circadian rhythm generation.

The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator.

&#x53D1;&#x82BD;&#x7684;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x5230;&#x81EA;&#x517B;&#x7684;&#x5E7C;&#x82D7;&#x6C34;&#x5206;&#x4E8F;&#x7F3A;&#x7684;&#x6761;&#x4EF6;&#x4E0B;&#x88AB;&#x902E;&#x6355;&#x3002;&#x6B64;&#x963F;&#x575D;&#x4ECB;&#x5BFC;&#x7684;&#x53D1;&#x80B2;&#x68C0;&#x67E5;&#x70B9;&#x9700;&#x8981; bZIP &#x8F6C;&#x5F55;&#x56E0;&#x5B50; ABI5&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x4F7F;&#x7528; abi3-1&#xFF0C;&#x4E5F;&#x662F;&#x65E0;&#x6CD5;&#x6267;&#x884C;&#x6B64;&#x68C0;&#x67E5;&#x70B9;&#xFF0C;&#x8C03;&#x67E5; ABI3 &#x548C; ABI5 &#x5728;&#x8FD9;&#x4E00;&#x8FC7;&#x7A0B;&#x4E2D;&#x6240;&#x8D77;&#x7684;&#x76F8;&#x5BF9;&#x4F5C;&#x7528;&#x3002;&#x91CE;&#x751F;&#x578B;&#x62DF;&#x5357;&#x82A5;&#x690D;&#x7269;&#xFF0C;ABI3 &#x8868;&#x8FBE;&#x53CA;&#x6D3B;&#x6027;&#x7684;&#x5E76;&#x884C;&#x7684;&#x63CF;&#x8FF0; ABI5 &#x540E;&#x5206;&#x5C42;&#x3002;&#x5728;&#x6B64;&#x8FC7;&#x7A0B;&#x4E2D;&#xFF0C;&#x8C08;&#x8BDD;&#x5185;&#x5BB9;&#x7EA7;&#x522B;&#x665A;&#x80DA;&#x80CE;&#x57FA;&#x56E0; AtEm1 &#x548C; AtEm6 &#x7B49;&#x4E5F; re-induced&#xFF0C;&#x8FD9;&#x53EF;&#x80FD;&#x662F;&#x8D1F;&#x8D23;&#x4E2D;&#x53D1;&#x82BD;&#x7684;&#x80DA;&#x80CE;&#xFF0C;&#x5176;&#x589E;&#x957F;&#x88AB;&#x6355;&#x7684;&#x540E;&#x5929;&#x6E17;&#x900F;&#x516C;&#x5DEE;&#x3002;ABI5 &#x8868;&#x8FBE;&#x5F0F;&#x5927;&#x5927;&#x51CF;&#x5C11; abi3-1 &#x7A81;&#x53D8;&#x4F53;&#xFF0C;&#x5176;&#x4E2D;&#x5177;&#x6709;&#x4F4E;&#x7684; AtEm1 &#x6216; AtEm6 &#x8868;&#x8FBE;&#x5F0F;&#x3002;&#x4EA4;&#x53C9;&#x4E92;&#x8865;&#x5B9E;&#x9A8C;&#x8868;&#x660E;&#x8BE5; 35S ABI5 &#x53EF;&#x4EE5;&#x8865;&#x5145; abi3-1&#xFF0C;&#x800C; 35S ABI3 &#x4E0D;&#x80FD;&#x8865;&#x5145; abi5-4&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E; ABI5 &#x4E0B;&#x6E38;&#x884C;&#x4E3A; ABI3 &#x91CD;&#x65B0;&#x6FC0;&#x6D3B;&#x540E;&#x671F;&#x80DA;&#x80CE;&#x53D1;&#x751F;&#x65B9;&#x6848;&#x5E76;&#x902E;&#x6355;&#x751F;&#x957F;&#x53D1;&#x82BD;&#x80DA;&#x80CE;&#x3002;&#x867D;&#x7136; ABI5 &#x59CB;&#x7EC8;&#x4F4D;&#x4E8E;&#x7EC6;&#x80DE;&#x6838;&#x4E2D;&#xFF0C;&#x67D3;&#x8272;&#x4F53;&#x6C89;&#x6DC0; &#xFF08;&#x82AF;&#x7247;&#xFF09; &#x5B9E;&#x9A8C;&#x63ED;&#x793A;&#x963F;&#x575D;&#x589E;&#x52A0; ABI5 &#x5165;&#x4F4F; AtEm6 &#x542F;&#x52A8;&#x5B50;&#x4E0A;&#x3002;

ABI5 &#x4E0B;&#x6E38;&#x7684;&#x884C;&#x4E3A; ABI3 &#x5728;&#x53D1;&#x82BD;&#x8FC7;&#x7A0B;&#x4E2D;&#x6267;&#x884C;&#x4F9D;&#x8D56;&#x4E8E;&#x963F;&#x575D;&#x5DDE;&#x7684;&#x589E;&#x957F;&#x902E;&#x6355;&#x3002;

The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination.

Le d&#xE9;veloppement d&#x26;#39;un embryon en germination dans un semis autotrophe est arr&#xEA;t&#xE9; en vertu des conditions de d&#xE9;ficit hydrique. Ce point de contr&#xF4;le ABA-m&#xE9;diation du d&#xE9;veloppement exige le facteur de transcription bZIP ABI5. Ici, nous avons utilis&#xE9; ABI3-1, qui est aussi incapable d&#x26;#39;ex&#xE9;cuter ce point de contr&#xF4;le, pour enqu&#xEA;ter sur le r&#xF4;le relatif des ABI3 et ABI5 dans ce processus. Chez les plantes d&#x26;#39;Arabidopsis de type sauvage, les ABI3 expression et l&#x26;#39;activit&#xE9; parall&#xE8;les celles d&#xE9;crites pour la stratification ABI5 suivant. Au cours de ce processus, les niveaux de transcription de g&#xE8;nes tels que l&#x26;#39;embryogen&#xE8;se fin AtEm1 et AtEm6 sont &#xE9;galement r&#xE9;-induite, ce qui pourrait &#xEA;tre responsable de la tol&#xE9;rance acquise osmotique chez les embryons germ&#xE9;s dont la croissance est arr&#xEA;t&#xE9;e. ABI5 expression est fortement r&#xE9;duite dans ABI3-1 mutants, ce qui a AtEm1 faible ou AtEm6 expression. Des exp&#xE9;riences de compl&#xE9;mentation ont montr&#xE9; que la Croix-35S-ABI5 pourrait compl&#xE9;ter ABI3-1, alors que la 35S-ABI3 ne peut pas compl&#xE9;ter ABI5-4. Ces r&#xE9;sultats indiquent que les actes en aval de la ABI5 ABI3 de r&#xE9;activer les programmes de l&#x26;#39;embryogen&#xE8;se tardive et d&#x26;#39;arr&#xEA;ter la croissance des embryons en germination. Bien que ABI5 est toujours situ&#xE9; dans le noyau, immunopr&#xE9;cipitation chromosomique (ChIP) exp&#xE9;riences ont r&#xE9;v&#xE9;l&#xE9; que l&#x26;#39;ABA augmente ABI5 d&#x26;#39;occupation sur le promoteur AtEm6.

ABI5 agit en aval de la ABI3 d&#x26;#39;ex&#xE9;cuter un arr&#xEA;t de la croissance ABA-d&#xE9;pendantes Lors de la germination

ABI5 Handlungen hinter ABI3 eine ABA-abh&#xE4;ngige Wachstum Verhaftung w&#xE4;hrend der Keimung auszuf&#xFC;hren.

Lo sviluppo di un embrione di germinazione in un semenzale autotrofi &egrave; arrestato in condizioni di deficit idrico. Questo checkpoint developmental ABA-mediata richiede il fattore di trascrizione del bZIP ABI5. Qui, abbiamo usato abi3-1, che &egrave; anche in grado di eseguire questo checkpoint, per indagare il ruolo relativo di ABI3 e ABI5 in questo processo. In piante di Arabidopsis wild-type, espressione ABI3 e attivit&agrave; parallela quelli descritti per ABI5 seguito di stratificazione. Durante questo processo, i livelli di trascrizione del tardo geni embriogenesi come AtEm1 e AtEm6 sono anche re-induced, che potrebbe essere responsabile per la tolleranza osmotica acquisita negli embrioni germinati, cui crescita &egrave; arrestato. ABI5 espressione &egrave; notevolmente ridotta in mutanti di abi3-1, che ha bassa espressione di AtEm1 o AtEm6. Croce complementazione esperimenti hanno mostrati che 35S-ABI5 potrebbe integrare abi3-1, mentre 35S-ABI3 non pu&ograve; integrare abi5-4. Questi risultati indicano che ABI5 agisce a valle di ABI3 per riattivare programmi embriogenesi tardiva e alla crescita di arresto di embrioni di germinazione. Anche se ABI5 &egrave; costantemente si trova nel nucleo, esperimenti di immunoprecipitazione cromosomiche (ChIP) ha rivelato che ABA aumenta ABI5 occupazione sul promotore AtEm6.

ABI5 atti a valle di ABI3 per eseguire un arresto di crescita ABA-dipendente durante la germinazione.

&#x72EC;&#x7ACB;&#x6804;&#x990A;&#x82D7;&#x306B;&#x767A;&#x82BD;&#x80DA;&#x306E;&#x958B;&#x767A;&#x304C;&#x6C34;&#x8D64;&#x5B57;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x902E;&#x6355;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;ABA-&#x4EF2;&#x4ECB;&#x306E;&#x767A;&#x9054;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x306F;&#x3001;bZIP&#x578B;&#x8EE2;&#x5199;&#x56E0;&#x5B50;ABI5&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3067;ABI3&#x3068;ABI5&#x306E;&#x76F8;&#x5BFE;&#x7684;&#x306A;&#x5F79;&#x5272;&#x3092;&#x8ABF;&#x67FB;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3082;&#x3001;&#x3053;&#x306E;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x3092;&#x5B9F;&#x884C;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;ABI3-1&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x307E;&#x3057;&#x305F;&#x3002;&#x91CE;&#x751F;&#x578B;&#x30B7;&#x30ED;&#x30A4;&#x30CC;&#x30CA;&#x30BA;&#x30CA;&#x690D;&#x7269;&#x3001;ABI3&#x306E;&#x767A;&#x73FE;&#x3068;&#x6D3B;&#x6027;&#x3092;&#x4E26;&#x5217;&#x306B;&#x305D;&#x308C;&#x3089;&#x306F;ABI5&#x3001;&#x6B21;&#x306E;&#x5C64;&#x5225;&#x5316;&#x306E;&#x305F;&#x3081;&#x306B;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x9593;&#x3001;&#x305D;&#x306E;&#x3088;&#x3046;&#x306A;AtEm1&#x3068;AtEm6&#x3068;&#x3057;&#x3066;&#x5F8C;&#x671F;&#x80DA;&#x767A;&#x751F;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8EE2;&#x5199;&#x30EC;&#x30D9;&#x30EB;&#x306F;&#x307E;&#x305F;&#x518D;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x3001;&#x6210;&#x9577;&#x304C;&#x902E;&#x6355;&#x3055;&#x308C;&#x3066;&#x767A;&#x82BD;&#x3057;&#x305F;&#x80DA;&#x3067;&#x53D6;&#x5F97;&#x3057;&#x305F;&#x6D78;&#x900F;&#x5727;&#x8010;&#x6027;&#x306E;&#x8CAC;&#x4EFB;&#x3067;&#x3042;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002; ABI5&#x5F0F;&#x306F;&#x975E;&#x5E38;&#x306B;&#x4F4E;&#x3044;AtEm1&#x307E;&#x305F;&#x306F;AtEm6&#x5F0F;&#x304C;&#x3042;&#x308A;ABI3-1&#x5909;&#x7570;&#x4F53;&#x3067;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3059;&#x3002;&#x30AF;&#x30ED;&#x30B9;&#x76F8;&#x88DC;&#x6027;&#x5B9F;&#x9A13;&#x3067;&#x306F;35S-ABI3&#x304C;abi5-4&#x3092;&#x88DC;&#x5B8C;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x306A;&#x3044;&#x306E;&#x306B;&#x5BFE;&#x3057;&#x3001;35S-ABI5&#x306F;&#x3001;ABI3-1&#x3092;&#x88DC;&#x5B8C;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;ABI3&#x306E;&#x4E0B;&#x6D41;ABI5&#x884C;&#x70BA;&#x304C;&#x9045;&#x3044;&#x80DA;&#x767A;&#x751F;&#x306E;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3092;&#x518D;&#x958B;&#x3059;&#x308B;&#x3068;&#x767A;&#x82BD;&#x80DA;&#x306E;&#x6210;&#x9577;&#x3092;&#x902E;&#x6355;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; ABI5&#x304C;&#x4E00;&#x8CAB;&#x3057;&#x3066;&#x6838;&#x5185;&#x306B;&#x4F4D;&#x7F6E;&#x3057;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x67D3;&#x8272;&#x4F53;&#x514D;&#x75AB;&#x6C88;&#x964D;&#xFF08;ChIP&#xFF09;&#x5B9F;&#x9A13;&#x306F;&#x3001;ABA&#x304C;AtEm6&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306B;ABI5&#x5360;&#x6709;&#x7387;&#x3092;&#x5897;&#x52A0;&#x3055;&#x305B;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002;

&#x767A;&#x82BD;&#x6642;&#x306E;ABA-&#x4F9D;&#x5B58;&#x6027;&#x5897;&#x6B96;&#x505C;&#x6B62;&#x3092;&#x5B9F;&#x884C;&#x3059;&#x308B;ABI3&#x306E;&#x4E0B;&#x6D41;ABI5&#x884C;&#x70BA;

Autotrophic &#xC885;&#xBB18;&#xC5D0; germinating &#xBC30;&#xC544;&#xC758; &#xAC1C;&#xBC1C; &#xBB3C; &#xC801;&#xC790;&#xC758; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xCCB4;&#xD3EC; &#xB429;&#xB2C8;&#xB2E4;. ABA &#xC911;&#xC7AC; &#xBC1C;&#xB2EC; &#xAC80;&#xC0AC;&#xC810;&#xC774; bZIP &#xC804;&#xC0AC; &#xC778;&#xC790; ABI5 &#xD544;&#xC694;&#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0;,&#xC774; &#xACFC;&#xC815;&#xC5D0;&#xC11C; ABI3 &#xBC0F; abi5&#xC758; &#xC0C1;&#xB300; &#xC5ED;&#xD560;&#xC744; &#xC870;&#xC0AC; &#xD558;&#xB294; &#xB610;&#xD55C;&#xC774; &#xAC80;&#xC0AC;&#xC810;&#xC744; &#xC2E4;&#xD589;&#xD560; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;, abi3-1&#xC744; &#xC0AC;&#xC6A9; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; Arabidopsis &#xC2DD;&#xBB3C;&#xC5D0;&#xC11C; ABI3 &#xD45C;&#xD604;&#xACFC; &#xD65C;&#xB3D9; &#xBCD1;&#xB82C; abi5&#xC5D0; &#xB300; &#xD55C; &#xC124;&#xBA85; &#xD558;&#xB294; &#xACC4;&#xCE35;&#xC5D0; &#xB530;&#xB77C;. &#xC774; &#xACFC;&#xC815; &#xD6C4;&#xBC18;&#xC758; AtEm1 &#xBC0F; AtEm6 &#xAC19;&#xC740; embryogenesis &#xC720;&#xC804;&#xC790;&#xB294; re-induced&#xB3C4;, &#xB300; &#xBCF8; &#xC218;&#xC900; &#xC788;&#xB294; &#xC138;&#xADE0;&#xB41C; &#xBC30;&#xC544;&#xC758; &#xC131;&#xC7A5; &#xCCB4;&#xD3EC;&#xC5D0; &#xC778;&#xC218; &#xC0BC; &#xD22C; &#xD5C8;&#xC6A9; &#xC624;&#xCC28;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. ABI5 &#xC2DD; &#xD06C;&#xAC8C; abi3 1 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC5D0; &#xB0AE;&#xC740; AtEm1 &#xB610;&#xB294; AtEm6 &#xC2DD;&#xC774; &#xC904;&#xC5B4;&#xB4ED;&#xB2C8;&#xB2E4;. &#xBCF4;&#xC644;&#xC131; &#xC2E4;&#xD5D8; &#xBCF4;&#xC5EC;&#xC900; &#xD06C;&#xB85C;&#xC2A4; 35S ABI3 abi5 4 &#xBCF4;&#xC644; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4; &#xBC18;&#xBA74; &#xADF8; 35S-ABI5 abi3-1, &#xBCF4;&#xC644; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; ABI5 &#xD6C4;&#xBC18; embryogenesis &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xD65C;&#xC131;&#xD654; &#xD558;&#xB824;&#xBA74; abi3&#xC758; &#xACE0; &#xCD9C; &#xC544; &#xBC30;&#xC544;&#xC758; &#xC131;&#xC7A5; &#xCCB4;&#xD3EC; &#xD558;&#xB958; &#xC5ED;&#xD560; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. ABI5 &#xD575;&#xC5D0; &#xC77C;&#xAD00; &#xB418; &#xAC8C; &#xC788;&#xB354;&#xB77C;&#xB3C4; &#xC5FC;&#xC0C9;&#xCCB4; immunoprecipitation (&#xCE69;) &#xC2E4;&#xD5D8; ABA &#xC99D;&#xAC00; ABI5 &#xC778; AtEm6 &#xBC1C;&#xAE30;&#xC778;&#xC5D0; &#xBC1D;&#xD614;&#xB2E4;.

&#xBC1C; &#xC544; &#xC911; ABA &#xC885;&#xC18D; &#xC131;&#xC7A5; &#xCCB4;&#xD3EC;&#xB97C; &#xC2E4;&#xD589; &#xD558;&#xB294; abi3&#xC758; ABI5 &#xD589;&#xC704; &#xD558;&#xB958;.

El desarrollo de un embri&#xF3;n en la germinaci&#xF3;n de las pl&#xE1;ntulas en un aut&#xF3;trofo es arrestado bajo condiciones de d&#xE9;ficit h&#xED;drico. Este puesto de control en el desarrollo mediada por ABA requiere que el factor de transcripci&#xF3;n bZIP ABI5. En este caso, hemos utilizado ABI3-1, que tambi&#xE9;n es incapaz de ejecutar este punto de control, para investigar el papel relativo de ABI3 y ABI5 en este proceso. En las plantas de Arabidopsis de tipo salvaje, la expresi&#xF3;n y actividad paralela ABI3 los descritos para ABI5 siguiente estratificaci&#xF3;n. Durante este proceso, los niveles de transcripci&#xF3;n de los genes de embriog&#xE9;nesis tard&#xED;a como AtEm1 AtEm6 y tambi&#xE9;n se re-inducida, lo que podr&#xED;a ser responsable de la tolerancia adquirida osm&#xF3;tica en embriones germinados cuyo crecimiento se detiene. ABI5 expresi&#xF3;n se reduce en gran medida en el ABI3-1 mutantes, que tiene baja AtEm1 o expresi&#xF3;n AtEm6. Experimentos de complementaci&#xF3;n de la Cruz puso de manifiesto que 35S-ABI5 podr&#xED;a complementar ABI3-1, mientras que el 35S-ABI3 no puede complementar abi5-4. Estos resultados indican que ABI5 actos intermedios de ABI3 para reactivar los programas finales embriog&#xE9;nesis y para detener el crecimiento de los embriones en germinaci&#xF3;n. Aunque ABI5 est&#xE1; constantemente localizado en el n&#xFA;cleo, inmunoprecipitaci&#xF3;n cromos&#xF3;mica (CHIP) experimentos revelaron que el ABA aumenta ABI5 de ocupaci&#xF3;n en el promotor AtEm6.

ABI5 Hechos aguas abajo de ABI3 para ejecutar una detenci&#xF3;n del crecimiento dependiente de ABA durante la germinaci&#xF3;n

Plants have evolved protective mechanisms to ensure their survival when threatened by adverse environmental conditions during their transition to autotrophic growth. During germination, there is a 2- to 3-d period during which a plant can execute growth arrest when challenged by water deficit. This postgermination developmental checkpoint is signaled by the stress hormone abscisic acid (ABA), which induces the expression of the bZIP transcription activator ABI5. The growth arrest efficiency depends on ABI5 levels, and abi5 mutants are ABA-insensitive and unable to execute the ABA-mediated growth arrest. Here we show that a novel ABI5-interacting protein, designated as AFP, can form high molecular weight (Mr) complexes with ABI5 in embryo-derived extracts. Like ABI5, ABI five binding protein (AFP) mRNA and protein levels are induced by ABA during seed germination. Two different afp mutant alleles (afp-1 and afp-2) are hypersensitive to ABA, whereas transgenic plants overexpressing AFP are resistant; in these plants, AFP and ABI5 protein levels are inversely correlated. Genetic analysis shows that abi5-4 is epistatic to afp-1, indicating the ABA hypersensitivity of afp mutants requires ABI5. Proteasome inhibitor studies show that ABI5 stability is regulated by ABA through ubiquitin-related events. When expressed together, AFP and ABI5 are colocalized in nuclear bodies, which also contain COP1, a RING motif protein. Our results suggest that AFP attenuates ABA signals by targeting ABI5 for ubiquitin-mediated degradation in nuclear bodies.

AFP is a novel negative regulator of ABA signaling that promotes ABI5 protein degradation.

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.

The abi1-1 mutation blocks ABA signaling downstream of cADPR action.

Embryogenesis in Arabidopsis results in mature, osmotolerant embryos within dry seeds. Late-embryogenesis programs involve the transcription factors ABI3 and ABI5, which are necessary for osmotolerance. ABI3 and ABI5 are degraded in seeds initiating germination, abolishing their protected state. However, during an early stage of germination, strong osmotic stresses, or ABA exposure maintain ABI3 and ABI5 expression, leading to growth arrest, and osmotolerance. Mild stress stimuli delay ABI3 and ABI5 disappearance, retarding germination but not preventing eventual closure of embryogenesis programs. PICKLE (PKL), a putative chromatin modifier, is necessary to repress ABI3 and ABI5 expression during germination in response to ABA. We show that pkl mutants display persistent high expression of ABI3 and ABI5 upon ABA stimulation. In turn, maintenance of ABI5 expression leads to hypersensitive germination responses to ABA in pkl seeds. We provide evidence that ABI3 and ABI5 are less associated with repressed chromatin in pkl mutants. Our results provide evidence that PKL-dependent repression of embryonic gene expression extends to late-embryogenesis genes and is associated with changes in chromatin. We suggest that effective closure of embryogenesis omostolerance programs during germination prevents excessive plant reactions to stress.

The role of chromatin-remodeling factor PKL in balancing osmotic stress responses during Arabidopsis seed germination.

Seed germination is antagonistically controlled by the phytohormones gibberellic acid (GA) and abscisic acid (ABA). GA promotes seed germination by enhancing the proteasome-mediated destruction of RGL2 (for RGA-LIKE2), a key DELLA factor repressing germination. By contrast, ABA blocks germination by inducing ABI5 (for ABA-INSENSITIVE5), a basic domain/leucine zipper transcription factor repressing germination. Decreased GA synthesis leads to an increase in endogenous ABA levels through a stabilized RGL2, a process that may involve XERICO, a RING-H2 zinc finger factor promoting ABA synthesis. In turn, increased endogenous ABA synthesis is necessary to elevate not only ABI5 RNA and protein levels but also, critically, those of RGL2. Increased ABI5 protein is ultimately responsible for preventing seed germination when GA levels are reduced. However, overexpression of ABI5 was not sufficient to repress germination, as ABI5 activity requires phosphorylation. The endogenous ABI5 phosphorylation and inhibition of germination could be recapitulated by the addition of a SnRK2 protein kinase to the ABI5 overexpression line. In sleepy1 mutant seeds, RGL2 overaccumulates; germination of these seeds can occur under conditions that produce low ABI5 expression. These data support the notion that ABI5 acts as the final common repressor of germination in response to changes in ABA and GA levels.

The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity.

Under the canopy, far-red (FR) light represses seed germination by inactivating phytochrome photoreceptors. This elicits a decrease in gibberellins (GA) levels and an increase in abscisic acid (ABA) levels. GA promotes germination by enhancing the proteasome-mediated destruction of DELLA repressors. ABA prevents germination by stimulating the expression of ABI repressors. How phytochromes elicit changes in hormone levels or how GA- and ABA-dependent signals are coordinated to repress germination remains poorly understood. We show that repression of germination by FR light involves stabilized DELLA factors GAI, RGA and RGL2 that stimulate endogenous ABA synthesis. In turn, ABA blocks germination through the transcription factor ABI3. The role of PIL5, a basic helix-loop-helix transcription factor stimulating GAI and RGA expression, is significant, provided GA synthesis is high enough; otherwise, high GAI and RGA protein levels persist to block germination. Under white light, GAI and RGA driven by the RGL2 promoter can substitute for RGL2 to promote ABA synthesis and repress germination, consistent with the recent findings with RGL2. The three DELLA factors inhibit testa rupture whereas ABI3 blocks endosperm rupture.

Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity.

Under unfavorable environmental conditions, the stress phytohormone ABA inhibits the developmental transition from an embryo in a dry seed into a young seedling. We developed a genetic screen to isolate Arabidopsis thaliana mutants whose early seedling development is resistant to ABA. Here, we report the identification of a recessive mutation in AUXIN RESISTANT1 (AUX1), encoding a cellular auxin influx carrier. Although auxin is a major morphogenesis hormone in plants, little is known about ABA-auxin interactions during early seedling growth. We show that aux1 and pin2 mutants are insensitive to ABA-dependent repression of embryonic axis (hypocotyl and radicle) elongation. Genetic and physiological experiments show that this involves auxin transport to the embryonic axis elongation zone, where ABA enhances the activity of an auxin-responsive promoter. We propose that ABA represses embryonic axis elongation by potentiating auxin signaling in its elongation zone. This involves repression of the AUXIN INDUCIBLE (Aux/IAA) gene AXR2/IAA7, encoding a key component of ABA- and auxin-dependent responses during postgerminative growth.

Abscisic acid represses growth of the Arabidopsis embryonic axis after germination by enhancing auxin signaling.

pkl mutant seed germination is hypersensitive to ABA treatment due to abnormally high and persistent ABI3 and ABI5 expression. PKL, a putative chromatin modifier, is instrumental to associate ABI3 and ABI5 with silent chromatin in response to ABA. Thus, PKL prevents exaggerated germination arrest responses by shutting off ABI3 and ABI5 expression in response to mild stresses.

Arabidopsis seed germination responses to osmotic stress involve the chromatin modifier PICKLE.

We recently reported that the DELLA factor RGL2 represses testa rupture in response to changes in ABA and GA levels. Here, we provide genetic evidence that this observation extends to RGL3, another DELLA factor whose function was not previously characterized. However, RGL3's repressive activity was seen only in an rgl2 genetic background. This may be explained by the observation that RGL3's mRNA levels are positively regulated by ABA and low GA but to a lesser extent than those of RGL2. This could ensure that RGL2's repressive activity dominates relative to that of RGL3 under most germination conditions.

The GA-signaling repressor RGL3 represses testa rupture in response to changes in GA and ABA levels.

The stress phytohormone ABA inhibits the developmental transition taking the mature embryo in the dry seed towards a young seedling. ABA also induces the accumulation of the basic leucine zipper (bZIP) transcription factor ABA-insensitive 5 (ABI5) which, apart from blocking endosperm rupture, also protects the embryo by stimulating the expression of late embryogenesis abundant (LEA) genes that conferred osmotolerance during seed maturation. It is unknown whether ABA recruits additional embryonic pathways to control early seedling growth and fitness. Here we identify gia3 (growth insensitive to ABA3), a recessive locus in Arabidopsis mediating cotyledon cellular maturation and ABA-dependent repression of cotyledon expansion and greening. Microarray studies showed that expression of the essential mid-embryogenesis gene Maternal Embryo Effect 26 (MEE26) is induced by ABA during early seedling growth in wild-type (WT) or abi5 plants but not in gia3 mutants. However, we also show that the GIA3 locus controls ABA-dependent gene expression responses that partially overlap with those controlled by ABI5. Thus, the gia3 locus identifies an additional arm of ABA signaling, distinct from that controlled by ABI5, which recruits MEE26 expression and maintains cotyledon embryonic identity. Fine mapping localized the gia3 locus within a 1 Mb interval of chromosome 3, containing a large DNA insertion of a duplicated region of chromosome 2. It remains unknown at present whether gia3 phenotypes are the result of single or multiple genetic alterations.

Identification of growth insensitive to ABA3 (gia3), a recessive mutation affecting ABA Signaling for the control of early post-germination growth in Arabidopsis thaliana.

Endosperm rupture and lateral root emergence are biological processes involving organ emergence out of cellular layers. We review here the main similarities and differences between these two developmental processes and discuss the possibility that both could be associated with auxin-dependent regulation of cell wall remodelling gene expression. We speculate that endosperm rupture may serve as a model system for the study of certain aspects of lateral root emergence.

Endosperm rupture as a model for lateral root emergence in Arabidopsis?

Seed dormancy is an ecologically important adaptive trait in plants whereby germination is repressed even under favorable germination conditions such as imbibition with water. In Arabidopsis and most plant species, dormancy absolutely requires an unidentified seed coat germination-repressive activity and constitutively higher abscisic acid (ABA) levels upon seed imbibition. The mechanisms underlying these processes and their possible relationship are incompletely understood. We developed a "seed coat bedding" assay monitoring the growth of dissected embryos cultured on a layer of seed coats, allowing combinatorial experiments using dormant, nondormant, and various genetically modified seed coat and embryonic materials. This assay, combined with direct ABA measurements, revealed that, upon imbibition, dormant coats, unlike nondormant coats, actively produce and release ABA to repress embryo germination, whatever the embryo origin, i.e., from dormant, nondormant, or never dormant aba seeds, unable to synthesize ABA. The persistent high ABA levels in imbibed dormant seeds requires the permanent expression of the DELLA gene RGL2, where it remains insensitive to gibberellins (GA) unlike in nondormant seeds. These findings present the seed coat as an organ actively controlling germination upon seed imbibition and provide a framework to investigate how environmental factors break seed dormancy.

A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.

Here, we describe a series of methods suitable for the reproducible and abundant isolation of total RNA, genomic DNA, and total protein from dry or imbibed Arabidopsis seeds. The resulting material is suitable for most standard molecular biology procedures.

Isolation of genetic material from Arabidopsis seeds.

Phytochromes phyB and phyA mediate a remarkable developmental switch whereby, early upon seed imbibition, canopy light prevents phyB-dependent germination, whereas later on, it stimulates phyA-dependent germination. Using a seed coat bedding assay where the growth of dissected embryos is monitored under the influence of dissected endosperm, allowing combinatorial use of mutant embryos and endosperm, we show that canopy light specifically inactivates phyB activity in the endosperm to override phyA-dependent signaling in the embryo. This interference involves abscisic acid (ABA) release from the endosperm and distinct spatial activities of phytochrome signaling components. Under the canopy, endospermic ABA opposes phyA signaling through the transcription factor (TF) ABI5, which shares with the TF PIF1 several target genes that negatively regulate germination in the embryo. ABI5 enhances the expression of phytochrome signaling genes PIF1, SOMNUS, GAI, and RGA, but also of ABA and gibberellic acid (GA) metabolic genes. Over time, weaker ABA-dependent responses eventually enable phyA-dependent germination, a distinct type of germination driven solely by embryonic growth.

Luis Lopez-Molina

Département de Botanique et de Biologie Végétale

Université de Genève

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Luis Lopez-Molina

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Département de Botanique et de Biologie Végétale

Université de Genève

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Département de Botanique et de Biologie Végétale