一作解读 | 小麦VRN1、FUL2和FUL3在小穗发育和穗部形态建成过程中起关键并冗余作用

小穗（spikelet）和小花（floret）是麦类作物花器官的基本结构，它们的发育直接影响到穗粒数和最终的籽粒产量。最近，Development杂志在线发表了Jorge Dubcovsky实验室（UC Davis）在小麦穗发育和形态建成方面的研究成果（doi:10.1242/dev.175398）。该研究系统解析了三个关键基因VRN1、FUL2和FUL3在小麦穗发育过程中的分子机理，并为将来提高小麦籽粒产量提供了直接证据。相关研究非常系统、全面，是一篇高质量的研究论文。我们有幸邀请到文章的一作--李成霞（Chenxia Li）老师进行解读，小麦研究联盟进行了翻译（由于我们在小麦穗发育方面的知识有所欠缺，如出现错误请参考李老师的英文解读）。

Wheat VRN1,FUL2andFUL3play critical and redundant roles in spikelet development and spike determinacy小麦VRN1、FUL2和FUL3在小穗发育和穗部形态建成过程中起关键并冗余作用

Chengxia Li^(#), Huiqiong Lin^(#), Andrew Chen, Meiyee Lau, Judy Jernstedt and Jorge Dubcovsky⁽*⁾

⁽*⁾：jdubcovsky@ucdavis.edu. Phone: 530 752 5159

The grass family (Poaceae) has approximately 10,000 species, including important food crops such as rice, maize, sorghum, barley and wheat.The flowers of these species are organized in a unique and diagnostic structure called spikelet, which is a compact inflorescence developing within the larger inflorescence. Spikelet is the basic unit of the grass inflorescence. Grass inflorescences have been described as a progressive acquisition of different meristem identities that begins with the transition of the vegetative shoot apical meristem (SAM) to an inflorescence meristem (IM). In wheat, the transition from vegetative SAM to IM is marked by the formation of a double-ridge structure, in which the lower leaf ridges are suppressed and the upper ridges acquire spikelet meristem (SM) identity andform spikelets. The number of spikelets per spike in wheat is determined by the number of lateral meristems formed before the transition of the IM into a SM toform the terminal spikelet. The growth of each wheatspikelet is indeterminate, with each SM initiating a variable number of floralmeristems (FM). The numbers of spikelets per spike and florets per spikelet determine the maximum number of grains per spike, therefore are important components of wheat grain yield potential.

禾本科（Poaceae）大约包含10,000个物种，其中包括水稻、玉米、高粱、小麦和大麦等重要的粮食作物。禾本科作物的花通过发育逐渐形成一种独特的结构--小穗，该结构属于一种在较大花序内发育的紧密花序。小穗是禾本科植物花器官的基本单位，其发育开始于营养生长的顶端分生组织（SAM）并逐渐向生殖生长的花序分生组织（IM）转变。在小麦中，从SAM到IM的转变以双脊结构的形成为特征，下部叶脊的发育受到抑制的同时上部脊继续发育形成小穗分生组织（SM）并逐渐发育成小穗。在这个过程中，花序分生组织（IM）逐渐转变为小穗分生组织（SM）并完成小穗的分化。在末端（最后一个）小穗分化形成之前所产生的侧向分生组织的数量决定了小麦中每个穗子的小穗数。小麦每个小穗分生组织（SM）可分化成可变数量的花分生组织（FM），因此小麦的小穗数并不固定。每个穗的小穗数和每个小穗的小花数决定了每个穗的最大穗粒数，是小麦籽粒产量潜力的重要组成部分。

In this study, we show that wheat MADS-box genes VRN1,FUL2andFUL3play critical and redundant roles in spikelet and spike development, and also affect flowering time and plant height. We combined loss-of-function mutants for the two homeologs ofVRN1,FUL2andFUL3 to generate double- and triple- mutants in tetraploid wheat. In thevrn1ful2ful3- triple mutant, the inflorescence meristem formed a normal double-ridge structure, however, itslateral meristems then proceeded to generate vegetative tillers subtended by leaves instead of spikelets. These results suggest an essential role of these three genes in the fate of the upper spikelet ridge and the suppression of the lower leaf ridge. Inflorescence meristems ofvrn1ful2ful3- andvrn1ful2- remained indeterminate, and singlevrn1- andful2-mutants showed delayed formation of the terminal spikelet and increased number of spikelets per spike. Moreover, theful2- mutant producedmore florets per spikelet, which together with a higher number of spikelets, resulted in a significant increase in the number of grains per spike in the field. Our results suggest that a better understanding of the mechanisms underlying wheat spikelet and spike development can inform future strategies toimprove grain yield in wheat.

我们的研究表明，小麦MADS-box基因VRN1、FUL2和FUL3在穗发育中起着关键和冗余的作用，并且还影响开花时间和植株高度。我们分别利用VRN1、FUL2和FUL3在AB亚组同时突变的功能丧失突变体，在四倍体小麦中创制了双重（4个基因）和三重（6个基因）突变体。在vrn1ful2ful3三重突变体中，花序分生组织能形成正常的双脊结构，但其侧向分生组织不能正常分化形成小穗，而是发育形成由叶子而不是小穗包围的营养分蘖。这个结果表明VRN1、FUL2和FUL3在促进上部小穗脊的正常发育和抑制下部叶脊的发育过程中具有重要作用。vrn1ful2ful3三重突变体和vrn1ful2双重突变体的花序分生组织发育异常，vrn1和ful2单突变体最后一个小穗原基分化较晚并导致了小穗数的增加。此外，ful2单突变体每个小穗还能产生更多的小花，并最终导致了穗粒数的增加。我们的研究结果阐明了小麦穗发育特别是小穗发育的分子机制，并为提高小麦穗粒数和产量提供了理论依据。

1. VRN1,FUL2 andFUL3 loss-of-function mutations reducestem elongation and delay flowering time1.VRN1、FUL2和FUL3功能丧失突变体减少了茎的伸长并延迟开花

Plants carrying only the ful3-mutation showed no significant reduction in stem length, but those carrying thevrn1-orful2-mutations were 20% and 14% shorter than the control, respectively (Fig. 1A). A three-way factorial ANOVA for stem length revealed highly significant effects for all three genes and significant synergistic interactions (Fig. 1C), indicating thatVRN1, FUL2 andFUL3 have redundant roles in the regulation of stem elongation, and that the effect of the individual genes is larger in the absence of the otherparalogs.

ful3单突变体株系的茎长或株高并没有明显变化，但vrn1或ful2单突变的株系分别比对照矮20％和14％（图1A）。三因素方差分析表明VRN1、FUL2和FUL3三个基因对茎杆长度具有显著影响并存在明显的协同作用（图1C），这表明三个基因在茎杆伸长的调节中作用冗余，并且在旁系同源基因缺失的情况下，单个基因的作用更大。

Functional redundancy among VRN1,FUL2 andFUL3 was also observed for heading time. Thevrn1- mutant headed 37.5 dlater than the control (Fig. 1D), but differences in heading time for theful2-,ful3-andful2ful3- mutants in the presence of the strongVrn-A1 allele were non-significant (Fig. 1E). For thevrn1ful2-andvrn1ful2ful3-mutants, it was not possible to determine heading times accurately because theyhad short stems and abnormal spikes that interfere with normal ear emergence. Instead, we determined the final number of leaves (Fig. 1B) and the timing of the transition between the vegetative and double-ridge stages (Fig. S3). Thevrn1- mutant had on average 14.4 leaves (59% > control, Fig. 1B), which was consistent with its later heading time (Fig. 1D). Similar leaf numbers were detected invrn1ful2-(14.3) andvrn1ful3-(14.9), but the triplevrn1ful2ful3-mutant had on average17.7 leaves (Fig. 1B), which was consistent with the 9 to 12 d delay in the transition between the vegetative SAM and the double-ridge stage relative to thevrn1-control (Fig. S3).

对抽穗时间来说，VRN1、FUL2和FUL3三个基因同样存在功能冗余。vrn1突变体的抽穗期比对照晚37.5天（图1D），但在强Vrn-A1等位基因存在下，ful2、ful3单突变体和ful2ful3双重突变体的抽穗时间差异并不显着（图1E）。对于vrn1ful2双重突变体和vrn1ful2ful3三重突变体来说，我们并不能准确地检测其抽穗时间，因为这类突变体的茎非常短、穗部发育异常并且不能形成正常叶耳。但我们仍然可以统计叶片的最终数量（图1B）以及小穗发育过程中从营养生长到生殖生长（双脊期）过渡时间（图S3）。vrn1单突变体平均具有14.4叶（比对照多59%，图1B），其抽穗时间也相应地延长（图1D）。在vrn1ful2双重突变体（14.3叶）和vrn1ful3双重突变体（14.9叶）中检测到相似的叶片数，但是三重突变体vrn1ful2ful3平均具有17.7叶（图1B），相应地三重突变体与vrn1单突变体相比开花时间也产生了9-12天的延迟（图S3）。

Fig. 1. VRN1、FUL2和FUL3对茎长、叶片数目和抽穗时间的影响

2. VRN1,FUL2andFUL3 play critical and redundant roles in spikelet development2.VRN1、FUL2和FUL3在小穗发育过程中发挥着关键和冗余的作用

Plants with individual vrn1-,ful2-andful3-mutations produced normal spikelets and flowers, butvrn1ful2-orvrn1ful2ful3-mutants had spike-like structures in which alllateral spikelets were replaced by leafy shoots (inflorescence tillers, Fig.2A-J). Removal of these inflorescence tillers revealed a thicker and shorter rachis with fewer internodes of variable length, but still retaining the characteristic alternating internode angles typical of a wild type rachis (Fig. 2B).

vrn1、ful2和ful3单突变的植物能产生正常的小穗和小花， vrn1ful2双重或vrn1ful2ful3三重突变体虽能产生类似小穗的结构，但其所有的侧面小穗均被叶状枝条替代（花序分蘖，图 2A-J）。去除这些花序分蘖后则显示出较粗和较短的穗轴，节间变少、节间长度也有不同程度的变化，但仍然保留了典型的野生型交替的穗轴结构（图2B）。

In vrn1ful2-, approximately 70% of the central inflorescence tillers had leafy glumes, lemmasand paleas and abnormal floral organs, whereas the rest were fully vegetative. Floral abnormalities included leafy lodicules, reduced number of anthers, anthers fused to ovaries, and multiple ovaries (Fig. 2E-G). After the first modified floret, meristems from these inflorescence tillers developed two to five true leaves before transitioning again to an IM generating lateral VMs(Fig. 2E). The presence of both floral organs and leaves suggests that the originating meristem had an intermediate identity between VM and SM before transitioning to an IM. In thevrn1ful2-double mutant the inflorescence tillers were subtended by bracts (Fig. 2C-D).

在vrn1ful2双突变体中，大约70％的中央花序分蘖具有叶状颖片、外稃和内稃以及异常的花器官结构，而其余部分则全为营养器官。花的异常包括叶片化的浆片、花药数量的减少、形成与子房融合的花药和多个子房等（图2E-G）。在第一个小花发育之后，来自花序分蘖的分生组织紧接着发育形成2至5个真叶，随后再继续发育形成侧面的顶端分生组织（图2E）。花器官和叶子的存在表明，早期的顶端分生组织在转变为花序分生组织之前既可以向营养生长分化也可以向生殖生长分化。在vrn1ful2双重突变体中，花序分蘖被苞片包围（图2C-D）。

In vrn1ful2ful3-, the lateral meristems generated inflorescence tillers that had no floralorgans, and that were subtended by leaves in the basal positions and bracts in more distal positions (Fig. 2H-J). The presence of well-developed axillary tillers in these basal inflorescence leaves (Fig. 2H, L19 and L20) marked the border of the spike-like structure, because no axillary tillers or developing buds were detected in the true leaves located below this border (Fig. 2H,L11-L18).

在vrn1ful2ful3三突变体中，侧生分生组织产生的花序分蘖没有花器官，而是在基部位置产生叶片组织，在另一端产生苞片（图2H-J）。在这些基部花序叶片组织中存在发育良好的腋生分蘖（图2H，L19和L20），代表着穗状结构的边界，因为在该边界下方的真叶中未检测到腋生分蘖或发育中的芽（图2H，L11-L18）。

Fig.2. vrn1ful2 双重突变体和vrn1ful2ful3 三重突变体的表型变异

Scanning Electron-Microscopy (SEM) images of the early developing inflorescences in the vrn1ful2-andvrn1ful2ful3-mutants revealed elongated double-ridge structures similar to those in Kronos (Fig. 3 A) orvrn1-(Fig. 3 C). Suppression of the lower leaf ridge was complete in Kronos (Fig. 3A) and invrn1-(Fig. 3D, red arrows), but was incomplete invrn1ful2-(Fig. 3B, E; yellow arrows), and even weaker invrn1ful2ful3-(Fig. 3C, F: green arrows). As a result of this change, inflorescence tillers were subtended by bracts invrn1ful2-(Fig. 2C-D) and by leaves invrn1ful2ful3-(Fig. 2H-I). The upper ridges (Fig. 3A-C, dots) transitioned into normal SMs invrn1-, with glume and lemma primordia (Fig. 3D, G), but looked like typical vegetative meristems invrn1ful2-andvrn1ful2ful3-(Fig. 3E-F, H-I).

通过扫描电子显微镜观察发现，vrn1ful2双重和vrn1ful2ful3三重突变体中在发育早期的花序中出现了类似于野生型Kronos（图3A）或vrn1单突变体（图3C）中的细长双脊结构。在Kronos（图3A）和vrn1单突变体中（图3D，红色箭头）下部叶脊的发育受到抑制，但在vrn1ful2双重突变体（图3B，E;黄色箭头）和vrn1ful2ful3三重突变体中（图3C，F：绿色箭头）叶脊的发育并没有受到正常抑制。因此，在vrn1ful2双重突变体中花序分蘖被苞片替代（图2C-D），而在vrn1ful2ful3三重突变体中花序分蘖被叶片替代（图2H-I）。在vrn1单突变体中，上部脊（图3A-C，红、黄或绿点）转变为正常的小穗分生组织（SM），并发育出颖片和外稃原基（图3D，G），但在vrn1ful2双重和vrn1ful2ful3三重突变体中则发育出了典型顶端分生组织（图 3E-F，HI）。

Fig.3. 不同突变体的扫描电镜观察

3. FUL2andVRN1haveredundant roles on spike determinacy and regulate the number of spikelets perspike3.FUL2和VRN1在穗形态建成过程中存在功能互补并调节小穗数

Normal wheat spikes are determinate, with the distal IM transitioning into a terminal spikelet after producing arelatively stable number of lateral meristems (Fig. 4A). In vrn1ful2-, by contrast, the IM was indeterminate (Fig. 4B) and continued to produce lateral meristems while growing conditions were favorable and eventually died without producing anyterminal structure. In theful2- background, one functional copy ofVRN1 inthe heterozygous state was sufficient to generate a determinate spike (Fig.S6D,ful2-/vrn-A1- vrn-B1), and the same was true for a single functional copy ofFUL2 in avrn1-background (Fig. S6K,vrn1-/ful2-A Ful2-B).

正常小麦在产生相对稳定数量的侧分生组织后，远端花序分生组织（distal IM）转变为末端（最后一个）小穗（图4A）。相比之下，在vrn1ful2双重突变体中，远端花序分生组织（IM）是不确定的（图4B），在合适的生长条件下可继续分化出侧向分生组织，并且最终死亡。在ful2单突变体背景中，杂合状态下VRN1的一个功能拷贝足以产生正常的幼穗（图S6D，ful2/vrn-A1 VRN-B1）；同样的，在vrn1单突变体背景中，杂合状态下FUL2的一个功能拷贝也能产生正常的幼穗（图S6K，vrn1/ful2-AFUL2-B）。

The individual vrn1-andful2-homozygous mutantsshowed a larger number of spikelets per spike than the control. This increasewas 58% in thevrn1-mutant (P< 0.0001, Fig. 4C) and 10% in theful2-mutant (P =0.0014, Fig 4D). Although no significant increases in the number of spikelets per spike were detected in theful3-mutant (P= 0.4096, Fig. 4E), two independent transgenic lines overexpressingFUL3 (Ubi::FUL3) showed an average reduction of 1.12 spikelet per spike relative to their non-transgenic sister lines (P = 0.0132 andP < 0.0001, Fig. S8A), which indicates thatFUL3can still play a role on the timing of the transition from IM to terminal spikelet.

与对照相比，vrn1或ful2单纯合突变体每个穗子的小穗数增多：vrn1单突变体（P <0.0001，图4C）的小穗数增加了58％，而ful2单突变体中的小穗数则增加了10％（P = 0.0014，图4D）。尽管在ful3单突变体中小穗数没有明显增加（P = 0.4096，图4E），但与非转基因姊妹系相比，过表达FUL3的两个独立转基因系（Ubi ::FUL3）的小穗数平均减少了1.12个（P = 0.0132和P <0.0001，图S8A），这表明FUL3仍然可以在花序分生组织（IM）转变到末端小穗的过程中发挥作用。

4. The ful2- mutant produces a highernumber of florets per spikelet andmore grains per spike in the field4.ful2单突变体小花数量变多并最终导致大田环境中穗粒数的增加

In addition to the higher number of spikelets per spike, the ful2-mutant produced a higher number of florets per spikelet than the Kronos control, an effect that was not observed forvrn1-(Fig. 2A) orful3-(Fig. S10A). In spite of some heterogeneity in the distribution of spikelets with extra florets among spikes, the differences between the control and theful2-mutants were significant at all spike positions (Fig. S10B).

与Kronos野生型对照相比，ful2单突变体除了小穗数量较多外，每个小穗的产生的小花数量也明显增加，这种变化在vrn1（图2A）或ful3单突变体中（图S10A）并未观察到。尽管具有额外小花的小穗在整个穗子中的分布存在一些异质性，但对照和ful2单突变体之间的差异在所有穗位置都是显着的（图S10B）。

Based on its positive effect on the number of florets perspikelet and spikelets per spike (and its small effect on heading time), we selected the ful2- mutant for evaluation in a replicated field experiment. Relative to the control, theful2-mutant produced 20% more spikelets per spike (P = 0.0002) and 9% more grains per spikelet (P= 0.05), which resulted in a 31% increase in the number of grains per spike (P= 0.0002, Fig. 4F). Although part of the positive effect on grain yield was offset by a 19% reduction in averagekernel weight (P= 0.0012), we observed a slight net increase of 6% in total grain weight per spike (P= 0.09, Fig. 4F). This negative correlation between grain number and grain weight suggests that in this particular genotype by environment combination grain yield was more limited by the “source” (produced and transported starch) than by the “sink” (number and size of grains).

基于ful2单突变体对小穗和小花数量的正面影响（对抽穗时间的影响较小），我们选择了该突变体用于田间试验中进行评估。于对照相比，ful2突变体每个穗的小穗数增加了20％的（P = 0.0002），每个小穗的穗粒数则增加了9％（P = 0.05），从而最终使穗粒数增加了31％（ P = 0.0002，图4F）。虽然千粒重减少了19％（P = 0.0012），但我们仍发现每个穗子的总粒重量净增加了6％（P = 0.09，图4F）。穗粒数和千粒重之间的这种负相关性表明，在这种特定的基因型中，籽粒最终产量更容易受“源”（生产和运输的淀粉）的限制，而不是穗粒数或籽粒大小的限制。

Fig.4. FUL2和VRN1在穗形态建成过程中存在功能互补并调节小穗数