Genetic dissection of ovule and embryo abortion in heat-stressed pea

豌豆植物和作物对高温和干旱等胁迫很敏感，很容易败育花朵和幼荚，从而降低产量。胚珠是豆荚中潜在的豌豆种子，在它被花粉授粉之前。在成功授粉后，每个受精的胚珠都含有一个发育成种子的小胚胎。花和种子的损失很可能是因为胚珠容易受到热害。虽然众所周知，授粉对高温和高温日子很挑剔，但我们对胚珠和幼胚在高温下的表现知之甚少。这个项目着眼于豌豆品种，并开发了测量隐藏在花中的胚珠的热胁迫的方法。我们首先使用两种传统方法对微小的豆荚和它们的胚珠进行处理，这些胚珠是在花开放四天后从植物上采集的。在这里，我们可以通过显微镜下的荧光染色来查看胚珠的健康状况，该染色突出了胚珠表面的伤口组织，称为胼胝质。我们还使用紫色染色，使我们能够看到胚珠内的胚珠和胚囊中正在发育的胚胎。我们计划测量在有和没有热应激的情况下，胚胎及其胚囊的胼胝质含量和大小。然后，我们的项目将使我们能够更早地看到胚珠的发育，并查看细胞核中的DNA是否受损。如果我们看到胚珠开始死亡，我们就可以确定流产是由DNA的热损伤引起的，还是母豆荚让胚珠挨饿造成的。我们还计划观察花粉、小豆荚、它们的胚珠和幼胚在有和没有热胁迫的情况下的基因表达。我们计划找到一种方法，利用来自RNA测序的基因表达来鉴定胚珠对逆境的敏感性，甚至在开花阶段更早。我们将使用我们的显微镜方法对18个豌豆品种进行测量，以找到能够在热浪中保持胚珠存活的最佳品种，这样它们就可以用来开发未来的豌豆品种，这些品种可以经受住我们更温暖的夏天。最终，我们的结果将确定最具胚珠耐热性的最佳品种，更好的是，添加一些新的实验方法，我们可以使用这些方法来简化和自动化寻找更好和更健壮的种子。这项研究是萨斯喀彻温省大学和萨斯喀彻温省Pulse Growers之间的行业合作伙伴关系，由联邦政府合作。

Pea plants and crops are sensitive to stress like heat and drought, and readily abort flowers and young pods, lowering yields. An ovule is a potential pea seed within a pod before it is fertilized by pollen. On successful pollination, each fertilized ovule contains a small embryo that develops into a seed. Flower and seed loss is likely because ovules are susceptible to heat damage. While pollination is known to be fussy about heat and high temperature days, we know far less about how ovules and young embryos fare in heat. This project looks at pea varieties and develops methods to measure heat stress on ovules hidden inside flowers. We are first using two traditional methods on tiny pods and their ovules, collected from plants four days after a flower is open. Here we can view ovule health with a fluorescent stain under the microscope that highlights wound tissue, called callose, on the ovule surface. We also use a purple stain that allows us to see inside an ovule to the developing embryo in an embryo sac. We plan to measure the amount of callose and size of the embryo and its embryo sac, with and without heat stress. Our project will then enable us to view ovules even earlier in development, and see if the DNA is damaged in cell nuclei. If we see ovules starting to die, we can determine if abortion was caused by heat damage to DNA or if the mother pod was starving the ovules. We also plan to look at gene expression of pollen, tiny pods, their ovules and young embryos, with and without heat stress. We plan to find a way using gene expression from RNA sequencing to identify ovule susceptibility to stress even earlier to the open flower stage. We will use our microscopy methods to measure 18 pea varieties to find the best varieties that can keep ovules alive in a heat wave, so they can be used to develop future pea varieties that can stand up to our warmer summers. Ultimately, our results will identify the best varieties with the most ovule heat-resistance, and even better, add some new experimental methods we can use to simplify and automate for finding better and robust seeds. This research is an industry partnership between the University of Saskatchewan and Saskatchewan Pulse Growers, with collaboration from the federal government.