Heat stress (HS) is becoming a major and constant threat to crop production and food security as global warming progresses. Consequently, understanding and improving crop tolerance to HS are currently among the most important targets in plant biology and breeding research (Langridge and Reynolds, 2021). Recent evidences suggest epigenetic mechanisms act as new layer of regulation to cope with HS (Ohama et al., 2017; Song et al., 2021). However, the specific regulatory module composed of epigenetic factor and transcription factor in establishing thermotolerance remains unclear. As a typical cool-season crop, wheat is vulnerable to HS, especially at the flowering and grain-filling stages (Kaur et al., 2019). Here, we report that TaHAG1 plays a pivotal role in thermotolerance by maintaining the photosynthetic stability in wheat.

TaHAG1 encodes a histone acetyltransferase, orthologous with Arabidopsis AtHAG1/GCN5 and rice OsHAG702 (Zheng et al., 2021). There are three TaHAG1 homoeologs in hexaploid wheat, whose deduced proteins contain conserved N-terminal HAT domain and C-terminal bromodomain (Figure 1a). TaHAG1 localizes in the nucleus (Figure 1b). Transcript of three TaHAG1 homoeologs was induced quickly under HS and then gradually increased in a similar pattern with the time of stress prolonging (Figure 1c).

To explore the function of TaHAG1 in the regulation of thermotolerance, we obtained transgenic wheat plants that either had TaHAG1 overexpressed (OE) or silenced via RNA interference (RNAi). The OE and RNAi plants showed similar phenotypes with wild-type Fielder under normal conditions (Figure 1d). Upon HS treatments, both Fielder and transgenic lines displayed a certain degree of wilting and growth inhibition. However, after recovery, the OE lines presented an obvious increase in fresh weight than Fielder, whereas the RNAi lines showed a pronounced wilting phenotype than Fielder and could not recover growth (Figure 1d,e). In addition, the OE lines kept more chlorophyll content and lower membrane damage than wild-types and RNAi lines after HS (Figure 1f,g).

To further validate the function of TaHAG1 in thermotolerance, the knockout lines of TaHAG1 were generated using CRISPR/Cas9 system. The mutation simultaneously in three homoeologs of TaHAG1 was lethal for wheat. Thus, the lines with simultaneous mutations at the two homoeologs TaHAG1-A and TaHAG1-B (1 bp insertion in TaHAG1-A and 25 bp deletion in TaHAG1-B, respectively) were selected for analysis. As expected, the KO lines exhibited stronger defects in thermotolerance as compared with Fielder plants under HS condition, including reduced fresh weight, more wilted leaves, lower chlorophyll content and severe membrane damage (Figure 1h–j).

We further examined the thermotolerance of transgenic lines with wild-types under field conditions. The plants were covered with manually constructed thermo-stress tents in grain-filling stage for 30 days, with uncovered individuals grown alongside as controls (Figure 1k,l). Under HS conditions, all OE lines exhibited much better fitness than the Fielder, including higher thousand kernel weight and grain width. In contrast, RNAi and KO lines exhibited severe inhibition compared with Fielder in term of these traits (Figure 1m,n). Meanwhile, no significant differences were found in agronomic phenotypes between OE, RNAi, KO and Fielder under control conditions, suggesting TaHAG1 contributes to thermotolerance without negative consequences for other developmental traits.

To explore the molecular basis of TaHAG1 in the regulation of thermotolerance, we performed RNA-sequencing in seedlings of Fielder and TaHAG1-OE after HS treatment. We reasoned that TaHAG1-regulated genes involved in thermotolerance would be enriched in the clusters where their expressions were up-regulated in OE lines compared with wild-type and induced by HS. Based on this, 663 genes were identified and considered as TaHAG1-regulated genes in response to HS. The most significantly enriched classes of these genes were those responsible for nucleosome organization, which was consistent with the role of TaHAG1 in histone modification (Figure 1o). Moreover, cellular component of plasma membrane and thylakoid was greatly enriched, indicating the membrane and photosynthetic system of TaHAG1-OE might be better adapted to HS treatment, which was further supported by lower electrolytic leakage and higher chlorophyll content in TaHAG1-OE plants. Notably, a series of typical genes reported to be involved in the regulation of photosynthetic apparatus were detected, such as TaG1 and TaPSBR1 that involved in stable assembly of PSII were up-regulated significantly in TaHAG1-OE lines than wild-type plants under HS (Figure 1p). These results are consistent with our observation that TaHAG1 enhanced maximal PSII quantum efficiency (Fv/Fm) in TaHAG1-OE lines under HS conditions (Figure 1q). Moreover, this suggested that elevated Fv/Fm may be part of the thermotolerance mechanism mediated by TaHAG1 overexpression.

As coactivators, the TaHAG1 is likely to be recruited to target promoters by direct or indirect interaction with DNA-binding regulators. To further explore regulatory mechanisms of TaHAG1, we performed yeast two-hybrid screening and identified one of TaHAG1 interactors as TaNACL, a NAC domain-containing protein. TaNACL is up-regulated after HS and encodes a nuclear protein with transcriptional activation activity (Figure 1r). We then corroborated the TaHAG1–TaNACL interaction using luciferase complementation assays, where coexpression of TaHAG1 with TaNACL generated strong luminescence signals that were not detected in the control pairs (Figure 1s). We also confirmed their interaction using Co-IP and BiFC assays (Figure 1t,u). The above findings that TaHAG1 facilitates TaG1 and TaPSBR1 expressions after HS, together with the interaction of TaHAG1 and TaNACL, promoted us to investigate whether TaG1 and TaPSBR1 are direct target of TaNACL. Transient transactivation assay showed that TaNACL was able to activate the expression of TaG1 and TaPSBR1 promoter-driven luciferase (LUC) reporters. Moreover, coexpression TaHAG1 with TaNACL led to a significant increase in TaG1 and TaPSBR1 promoter activation compared with the expression of each single effector (Figure 1v). We also conducted electrophoretic mobility shift assays to confirm whether TaNACL directly binds to TaG1 and TaPSBR1 regulatory regions using recombinant protein TaNACL-GST. It was shown that TaNACL physically bound to the biotin-labelled TaG1 and TaPSBR1 promoters in a CACG motif-dependent manner, and the binding was competed by unlabelled wild-type probes but not mutated probes (Figure 1w,x).

Together, our results demonstrate that TaHAG1 regulates the transcription of TaG1 and TaPSBR1 through interacting with TaNACL to enhance thermotolerance in wheat (Figure 1y). This study provides a potential approach to improve wheat thermotolerance by increasing TaHAG1 expression, without observable penalty on plant growth. The regulatory factors involved in thermotolerance identified in this study could also be of great value for genetic improvement in wheat and in other crops.

随着全球变暖的加剧，热应激 (HS) 正在成为对作物生产和粮食安全的主要且持续的威胁。因此，了解和提高作物对 HS 的耐受性是目前植物生物学和育种研究中最重要的目标之一（Langridge 和 Reynolds，  2021 年）。最近的证据表明，表观遗传机制作为应对 HS 的新调控层（Ohama等人，  2017 年；Song等人，  2021 年）。然而，由表观遗传因子和转录因子组成的特定调控模块在建立耐热性方面仍不清楚。作为典型的冷季作物，小麦易受 HS 的影响，尤其是在开花和灌浆阶段（Kaur等人，  2019 年）。在这里，我们报告 TaHAG1 通过维持小麦的光合作用稳定性在耐热性中起关键作用。

TaHAG1 编码组蛋白乙酰转移酶，与拟南芥AtHAG1/GCN5 和水稻 OsHAG702 直系同源（Zheng et al .,  2021）。六倍体小麦中存在三个TaHAG1同源物，其推断的蛋白质包含保守的 N 端 HAT 结构域和 C 端溴结构域（图 1a）。TaHAG1 定位在细胞核中（图 1b）。三个 TaHAG1 同源物的转录物在 HS 下迅速诱导，然后随着应力延长的时间以类似的模式逐渐增加（图 1c）。

为了探索TaHAG1在调节耐热性中的功能，我们获得了TaHAG1过表达 (OE) 或通过 RNA 干扰 (RNAi) 沉默的转基因小麦植物。OE 和 RNAi 植物在正常条件下显示出与野生型 Fielder 相似的表型（图 1d）。在 HS 处理后，Fielder 和转基因品系均表现出一定程度的萎蔫和生长抑制。然而，恢复后，OE系比Fielder的鲜重明显增加，而RNAi系比Fielder表现出明显的萎蔫表型，无法恢复生长（图1d，e）。此外，与野生型和 HS 后的 RNAi 系相比，OE 系保留了更多的叶绿素含量和更低的膜损伤（图 1f，g）。

为了进一步验证TaHAG1在耐热性中的功能，使用 CRISPR/Cas9 系统生成了TaHAG1的敲除系。TaHAG1的三个同源物同时发生的突变对小麦是致命的。因此，选择在两个同源物 TaHAG1-A和TaHAG1-B处具有同时突变的系（分别在TaHAG1-A中插入 1 bp和在TaHAG1-B中删除 25 bp ）进行分析。正如预期的那样，与 HS 条件下的 Fielder 植物相比，KO 系在耐热性方面表现出更强的缺陷，包括鲜重减少、叶片枯萎、叶绿素含量降低和严重的膜损伤（图 1h-j）。

我们进一步检查了野生型转基因品系在田间条件下的耐热性。在谷物灌浆阶段，植物被人工建造的热应力帐篷覆盖 30 天，未覆盖的个体作为对照生长（图 1k，l）。在 HS 条件下，所有 OE 品系都表现出比 Fielder 更好的适应度，包括更高的千粒重和粒宽。相比之下，与 Fielder 相比，RNAi 和 KO 系在这些性状方面表现出严重的抑制作用（图 1m，n）。同时，在对照条件下，OE、RNAi、KO 和 Fielder 之间的农艺表型没有显着差异，这表明 TaHAG1 有助于耐热性，而不会对其他发育性状产生负面影响。

为了探索 TaHAG1 在耐热性调节中的分子基础，我们对经过 HS 处理的 Fielder 和TaHAG1 -OE 幼苗进行了 RNA 测序。我们推断，与野生型相比，与野生型相比，在 OE 系中它们的表达上调并由 HS 诱导的簇中会富集与耐热性有关的 TaHAG1 调节基因。基于此，663 个基因被鉴定并被认为是响应 HS 的 TaHAG1 调节基因。这些基因中最显着富集的类别是那些负责核小体组织的基因，这与 TaHAG1 在组蛋白修饰中的作用一致（图 1o）。此外，质膜和类囊体的细胞成分大大丰富，表明TaHAG1的膜和光合系统-OE 可能更好地适应 HS 处理，这进一步得到了TaHAG1 -OE 植物中较低的电解泄漏和较高的叶绿素含量的支持。值得注意的是，在 HS 下， TaHAG1 -OE 系中检测到一系列参与光合装置调控的典型基因，如参与 PSII 稳定组装的TaG1和TaPSBR1比野生型植物显着上调。图 1p)。这些结果与我们的观察结果一致，即 TaHAG1在 HS 条件下增强了TaHAG1 -OE 线中的最大 PSII 量子效率（Fv/Fm）（图 1q）。此外，这表明升高的 Fv/Fm 可能是由TaHAG1过表达。

作为共激活剂，TaHAG1 很可能通过与 DNA 结合调节剂的直接或间接相互作用被招募到靶向启动子。为了进一步探索 TaHAG1 的调控机制，我们进行了酵母双杂交筛选，并将 TaHAG1 相互作用物之一鉴定为 TaNACL，一种含有 NAC 结构域的蛋白质。TaNACL在 HS 后上调并编码具有转录激活活性的核蛋白（图 1r）。然后，我们使用荧光素酶互补测定证实了 TaHAG1-TaNACL 相互作用，其中 TaHAG1 与 TaNACL 的共表达产生了在对照对中未检测到的强发光信号（图 1s）。我们还使用 Co-IP 和 BiFC 分析证实了它们的相互作用（图 1t，u）。上述发现 TaHAG1 促进TaG1和HS 后TaPSBR1的表达，以及 TaHAG1 和 TaNACL 的相互作用，促使我们研究TaG1和TaPSBR1是否是 TaNACL的直接靶标。瞬时反式激活测定表明，TaNACL 能够激活TaG1和TaPSBR1启动子驱动的荧光素酶 (LUC) 报告基因的表达。此外，与每个单一效应子的表达相比，TaHAG1 与 TaNACL 的共表达导致TaG1和TaPSBR1启动子激活显着增加（图 1v）。我们还进行了电泳迁移率变动分析以确认 TaNACL 是否直接与TaG1和TaPSBR1结合使用重组蛋白 TaNACL-GST 的调节区。结果表明，TaNACL 以 CACG 基序依赖性方式与生物素标记的TaG1和TaPSBR1启动子物理结合，并且结合由未标记的野生型探针而不是突变探针竞争（图 1w，x）。

总之，我们的结果表明，TaHAG1 通过与 TaNACL 相互作用来调节TaG1和TaPSBR1的转录，从而增强小麦的耐热性（图 1y）。该研究提供了一种通过增加TaHAG1表达来改善小麦耐热性的潜在方法，而不会对植物生长造成明显的影响。本研究中确定的涉及耐热性的调节因素也可能对小麦和其他作物的遗传改良具有重要价值。