Panicle architecture is a key determinant of grain yield in cereals, but the mechanisms governing panicle morphogenesis and organ development remain elusive. Here, we have identified a quantitative trait locus (qPA1) associated with panicle architecture using chromosome segment substitution lines from parents Nipponbare and 9311. The panicle length, branch number and grain number of Nipponbare were significantly higher than CSSL-9. Through map-based cloning and complementation tests, we confirmed that qPA1 was identical to SD1 (Semi Dwarf1), which encodes a gibberellin 20-oxidase enzyme participating in gibberellic acid (GA) biosynthesis. Transcript analysis revealed that SD1 was widely expressed during early panicle development. Analysis of sd1/osga20ox2 and gnp1/ osga20ox1 single and double mutants revealed that the two paralogous enzymes have non-redundant functions during panicle development, likely due to differences in spatiotemporal expression; GNP1 expression under control of the SD1 promoter could rescue the sd1 phenotype. The DELLA protein SLR1, a component of the GA signalling pathway, accumulated more highly in sd1 plants. We have demonstrated that SLR1 physically interacts with the meristem identity class I KNOTTED1-LIKE HOMEOBOX (KNOX) protein OSH1 to repress OSH1-mediated activation of downstream genes related to panicle development, providing a mechanistic link between gibberellin and panicle architecture morphogenesis.
20世紀(jì)中期，半矮稈水稻和小麥品種開始被大面積推廣使用，有效地解決了“高產(chǎn)和倒伏”這對(duì)相互制約的矛盾，使得作物產(chǎn)量達(dá)到歷史新高，這便是著名的“綠色革命”。隨著分子生物學(xué)的發(fā)展，科學(xué)家們?cè)诤髞?lái)的研究中逐漸認(rèn)識(shí)到這次“綠色革命”與赤霉素有關(guān)，并通過(guò)圖位克隆的方法克隆到了引起水稻半矮化的關(guān)鍵基因SD1（Semi-Dwarf1），編碼赤霉素合成途徑一個(gè)GA20-2氧化酶。由此，SD1也被稱為“綠色革命”基因。
本研究，作者利用來(lái)自親本日本晴和9311的染色體片段置換系鑒定了一個(gè)與穗部構(gòu)型相關(guān)的數(shù)量性狀基因座qPA1(QTL for panicle architecture on chromosome 1)。作者發(fā)現(xiàn)水稻染色體片段置換系CSSL-9的穗長(zhǎng)、分枝數(shù)和籽粒數(shù)都顯著低于日本晴。通過(guò)圖位克隆和互補(bǔ)實(shí)驗(yàn)，證實(shí)了qPA1與SD1完全相同。進(jìn)一步，通過(guò)CRISPR/Cas9用于敲除日本晴和Kasalath品系的SD1，SD1純合突變體的穗長(zhǎng)降低8~21%，一次分枝數(shù)減少18~26%，二次分枝數(shù)減少，每穗總粒數(shù)減少29~41%。這些結(jié)果表明，SD1功能的喪失導(dǎo)致了更小、更少的分枝穗，從而在兩個(gè)水稻品種內(nèi)導(dǎo)致產(chǎn)量下降。同時(shí)，過(guò)表達(dá)SD1，日本晴轉(zhuǎn)基因植株產(chǎn)生了一個(gè)更大的穗部，表明SD1對(duì)穗部結(jié)構(gòu)起著重要的的調(diào)控作用。組織原位雜交也表明了，SD1基因在軸分生組織、一次和二次分生組織以及小穗分生組織中廣泛表達(dá)。
進(jìn)化樹分析和表型分析表明，GA20ox1和GA20ox2可能存在著功能冗余，因此，作者對(duì)sd1/osga20ox2和gnp1/osga20ox1單雙突變體的分析表明，在穗部發(fā)育過(guò)程中具有非冗余功能，GNP1和SD1獨(dú)立起著調(diào)節(jié)穗部發(fā)育的作用，并導(dǎo)致GA的劑量效應(yīng)來(lái)影響植物發(fā)育。
機(jī)制分析方面，與野生型植株相比，sd1中水稻DELLA蛋白SLR1的含量顯著增加，進(jìn)一步揭示了GA信號(hào)在穗部發(fā)育中的關(guān)鍵作用。酵母雙雜、分裂熒光素酶和雙分子熒光互補(bǔ)(BIFC）證實(shí)，SLR1與KNOX蛋白OSH1可互作。為了研究SLR1是否能抑制OSH1的基因激活，Y1H和熒光素酶試驗(yàn)證實(shí)OSH1可以直接與ASP1啟動(dòng)子結(jié)合，并且SLR1和OSH1的共表達(dá)降低了ASP1的表達(dá)。
總之，該研究表明SD1是決定穗構(gòu)型的正調(diào)節(jié)因子。DELLA蛋白(SLR1)可以通過(guò)物理作用破壞KNOX蛋白OSH1對(duì)下游基因的激活。當(dāng)SD1功能喪失時(shí)，累積的SLR1與KNOX-1類蛋白相互作用，抑制KNOX-1介導(dǎo)的下游基因的激活。當(dāng)SD1具有正常功能時(shí)，DDELLA蛋白減少，不能與KNOX-1蛋白相互作用，促進(jìn)下游基因表達(dá)，從而誘導(dǎo)GA調(diào)控穗部發(fā)育。該研究揭示了赤霉素與穗構(gòu)型形態(tài)發(fā)生之間的新調(diào)控機(jī)制。