Flavonoids are ubiquitous in terrestrial plants with important physiological functions. The in planta flavonoid profile depends on the activities of different biosynthesis enzymes (Figure 1a). Flavanone 3‐hydroxylase (F3H) is a key enzyme channelling carbon flow towards the production of 3‐hydroxylated flavonoids, including flavonols and anthocyanidins. In Poaceae, F3H‐encoding genes are generally inactive in vegetative tissues which accumulate flavone derivatives as the predominant flavonoid metabolites. Meanwhile, sorghum produces 3‐deoxyanthocyanidins and flavones as phytoalexins for defence against pathogens such as Colletotrichum sublineola, the causal agent of anthracnose. The occurrences of 3‐hydroxylated flavonoids in sorghum are generally not well known. Only in some cultivars, anthocyanin pigments accumulate in mesocotyls of seedlings upon illumination following the activation of F3H and other anthocyanidin biosynthesis genes.

Here, we transformed the sorghum inbred line Tx430 with the coding region of SbF3H1 (Sb06g031790) under the control of a maize ubiquitin1 (ubi1) promoter for flavonoid profiling and disease phenotyping. Two independent transformants (OE3 and OE4) were selected for characterizations in T₃ generation. SbF3H1 expression was detected in different tissues of the OE lines, but only in spikelets of Tx430 control (Figure 1b). Both OE lines showed no observable phenotypes from seedling to ripening stages. However, seed numbers per head declined by 55.6% and 18.8% in OE3 and OE4, respectively, when compared to Tx430 control. This is likely due to subtle pollen defects; slight reductions in percentage of viable pollen and pollen germination rate were found in OE3 and both lines, respectively (Figure 1c). In contrast, the grain yield component of 100‐seed weight showed a 10% increase in OE3 but remained unchanged in OE4.

Acid‐hydrolysed extracts of different tissues were prepared for LC‐MS/MS analysis. The flavonols kaempferol, quercetin and isorhamnetin were detected in all spikelet extracts analysed, while isorhamnetin accumulated in slightly higher amount in OE3 than Tx430 control (Figure 1d). Hence, overexpressing SbF3H1 did not have a substantial impact on flavonol production in tissues with endogenous F3H activities. The presence of high levels of flavonol derivatives in spikelets of wild‐type sorghum suggests a potentially important role for male fertility as demonstrated in other plant species. For instance, flavonols function as antioxidants to regulate reactive oxygen species (ROS) homeostasis during pollen tube elongation in tomato (Muhlemann et al., 2018).

In flag leaf extracts, kaempferol, isorhamnetin and syringetin were identified in both transgenic lines, but none of these flavonols were found in Tx430 (Figure 1e). It is worth‐noting that syringetin (3′,5′‐O‐dimethylated flavonol) is absent from flavonol‐producing spikelets of Tx430 control. Formation of syringetin requires flavonoid 3′,5′‐hydroxylase (F3′5′H) activities to generate 3′,5′‐hydroxyl groups for O‐methylation. In some dicots, F3′5′Hs belonging to the P450 CYP75A subfamily are responsible for 5′‐hydroxylation of different flavonoid substrates (Seitz et al., 2006). Interestingly, a chrysoeriol‐specific 5′‐hydroxylase belongs to CYP75B subfamily is involved in tricin (a 3′,5′‐O‐dimethylated flavone) biosynthesis in grasses (Lam et al., 2019). Whether a CYP75A F3′5′H or a CYP75B flavonol‐specific 5′‐hydroxylase is required for syringetin biosynthesis in leaf tissues of transgenic SbF3H1 lines remains to be elucidated.

Flavonol synthase (FLS) is a downstream enzyme of F3H during flavonol biosynthesis. The first monocot FLS‐encoding gene functionally characterized was maize ZmFLS1 with a tandemly duplicated gene ZmFLS2 (Falcone Ferreyra et al., 2012). Both genes are constitutively expressed in most maize tissues and can be further induced by UV‐B treatment. Sorghum SbFLS1 is a highly conserved homolog of ZmFLSs (>96% sequence identity). Similarly, SbFLS1 is expressed in all tissues examined in Tx430 control (Figure 1b), suggesting that SbF3H1 expression may modulate flavonol production. Hence, flavonol derivatives were detected in spikelet but not in other tissues which lack SbF3H1 expression in wild‐type sorghum.

Flavone derivatives are the prevalent flavonoid metabolites in grass biomass. Consistently, apigenin, chrysoeriol and tricin were detected in spikelet samples, while apigenin and tricin were slightly increased and reduced in the OE lines, respectively (Figure 1d). Meanwhile, apigenin, luteolin, chrysoeriol and tricin were found in flag leaf samples. Compared with Tx430 control, level of apigenin was reduced in OE3 (Figure 1e).

Sorghum bran contains substantial amounts of 3‐deoxyanthocyanidins (Awika et al., 2004). Consistently, apigeninidin and luteolinidin were detected in Tx430 control whole seed extracts (Figure 1f). Interestingly, anthocyanidins (pelargonidin and cyanidin), besides flavonols, were detected in seed of both transgenic lines as 3‐hydroxylated flavonoids. Anthocyanidins and 3‐deoxyanthocyanidins are structurally similar except for the C3‐hydroxylation. As the endogenous 3‐deoxyanthocyanidin branch pathway is active in seeds, some enzymes may accept 3‐hydroxylated substrates for anthocyanidin biosynthesis. For example, two dihydroflavonol reductases (SbDFR1 and SbDFR3) could reduce dihydroflavonols and flavanones to produce the immediate precursors of anthocyanidins and 3‐deoxyanthocyanidins, respectively (Liu et al., 2010).

The elongated mesocotyl system was further used to investigate flavonoid profiles and disease phenotypes in sorghum seedlings following C. sublineola infection. LC‐MS/MS analysis revealed the accumulation of flavonol derivatives and cyanidin in inoculated mesocotyls of transgenic seedlings but not in those of Tx430 (Figure 1g). Meanwhile, accumulation of 3‐deoxyflavonoid phytoalexins (3‐deoxyanthocyainidin and flavones) was reduced in transgenic seedlings compared with Tx430 seedlings. Although flavonoid biosynthesis is activated considerably after infection (primarily through chalcone synthase induction), SbF3H1 is apparently diverting some of the carbon flow towards 3‐hydroxylated flavonoid production.

Expression of flavone biosynthesis genes [flavone synthase (SbFNSII; Sb02g000220), apigenin 3ʹ‐hydroxylase/chrysoeriol 5ʹ‐hydroxylase (SbA3ʹH/C5ʹH; Sb09g022480) and flavonoid O‐methyltransferase (SbFOMT; Sb07g003860)] was examined in spikelets, flag leaf and infected mesocotyls. No significant changes in gene expression levels were found between Tx430 and both transgenic lines, except a slight increase in the expression of SbFOMT was detected in flag leaf of OE3 (data not shown). Meanwhile, expression of 3‐deoxyanthocanidin biosynthesis genes [flavanone 4‐reductase (SbFNR; Sb06g029550) and SbDFR3 (Sb04g004290)] was not altered in infected mesocotyls of the OE lines. These results suggest that overexpressing SbF3H1 did not result in major alterations in expressions of genes in other flavonoid branch pathways.

For inoculated mesocotyls, disease severity was examined 6 days post‐inoculation as described (Schnippenkoetter et al., 2017). Occurrences of strong symptoms in Tx430 mesocotyls were 64% more compared with both transgenic mesocotyls (Figure 1h,i). By contrast, there were more transgenic seedlings with no disease symptoms than Tx430 control. Hence, SbF3H1 overexpression resulted in reduced susceptibility to C. sublineola albeit lower amounts of 3‐deoxyflavonoid phytoalexins were produced. Because sorghum pathogens are less likely to encounter flavonol and cyanidin derivatives in vegetative tissues of wild‐type sorghum, there may not be existing detoxification mechanisms in the fungus. In addition, these 3‐hydroxylated flavonoids may function synergistically with 3‐deoxyflavonoid phytoalexins to improve the overall antifungal capacity in the SbF3H1‐overexpressing plants.

Hydrogen peroxide and superoxide ion were analysed in sorghum mesocotyls by diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining, respectively (Figure 1j). In uninoculated seedlings, there was little or no DAB and NBT staining. Brownish DAB lesions were evident in Tx430 control mesocotyls 24 h post‐inoculation but barely observable in the transgenic mesocotyls. Similarly, NBT staining was more intense and widespread in infected Tx430 seedlings compared with infected transgenic seedlings. Overall, pathogen‐inducible ROS production is alleviated in the SbF3H1‐overexpressing lines. As flavonol derivatives are potent antioxidants (Heim et al., 2002), they may scavenge some ROS in the infected transgenic seedlings.

Previously, transgenic F3H overexpression was performed in tobacco which naturally accumulates 3‐hydroxylated flavonoids due to endogenous F3H activities, leading to enhanced accumulation of flavonols and flavan‐3‐ols without vigorous changes in flavonoid profile (Song et al., 2016). By contrast, we overexpressed SbF3H1 in sorghum which is deficient in 3‐hydroxylated flavonoids in vegetative tissues, resulting in partial re‐direction of carbon flow towards 3‐hydroxylated flavonoid production, hence increasing the complexity of flavonoid‐derived metabolites in different tissues. The enriched flavonoid profile may enhance defence response and improve nutraceutical values of sorghum grain/bran. In particular, syringetin derivatives, which are novel flavonoid metabolites not identified in sorghum previously, showed a range of health beneficial properties (Hsu et al., 2009). Considering seed yield reduction in the overexpression lines, the use of pathogen‐inducible and tissue‐specific promoters should be explored for driving targeted SbF3H1 expression without compromising grain productivity.

类黄酮在陆生植物中普遍存在，具有重要的生理功能。的在植物中类黄酮分布取决于不同生物合成的酶（图1a）的活动。黄烷酮3-羟化酶（F3H）是引导碳流向3-羟化类黄酮（包括黄酮醇和花色素苷）生成的关键酶。在禾本科中，F3H编码基因通常在营养组织中失活，而营养组织中积累的黄酮衍生物是主要的类黄酮代谢产物。同时，高粱会产生3-脱氧花青素和黄酮作为植物抗毒素，以抵御病原菌，例如炭疽菌，炭疽病的病因。高粱中3-羟基化类黄酮的存在通常是未知的。仅在某些品种中，在激活F3H和其他花色素苷生物合成基因后，光照后，花色素苷色素会在幼苗的中胚轴中积累。

在这里，我们在玉米泛素1（ubi1）启动子的控制下，用SbF3H1（Sb06g031790）的编码区转化了高粱自交系Tx430，以进行类黄酮分析和疾病表型鉴定。选择了两个独立的转化子（OE3和OE4）用于表征T ₃世代。SbF3H1在OE系的不同组织中检测到表达，但仅在Tx430对照的小穗中检测到（图1b）。从幼苗到成熟阶段，这两个OE品系都没有观察到表型。但是，与Tx430对照相比，OE3和OE4的人均种子数分别下降了55.6％和18.8％。这可能是由于微弱的花粉缺陷造成的。在OE3和两个品系中，分别发现存活花粉的百分比和花粉的发芽率略有下降（图1c）。相反，100粒重的谷物产量组成部分显示OE3增加10％，但OE4保持不变。

准备了不同组织的酸水解提取物用于LC-MS / MS分析。在所有分析的小穗提取物中都检测到黄酮醇山emp酚，槲皮素和异鼠李素，而异鼠李素在OE3中的蓄积量比Tx430对照略高（图1d）。因此，过表达的SbF3H1对具有内源性F3H活性的组织中的黄酮醇产生没有实质性影响。在野生型高粱的小穗中存在高含量的黄酮醇衍生物，这暗示了雄性育性的潜在重要作用，正如其他植物物种所证明的那样。例如，黄酮醇在番茄花粉管伸长过程中起抗氧化剂的作用，以调节活性氧（ROS）的稳态（Muhlemann et al。，2018）。

在旗叶提取物中，在两个转基因品系中均鉴定出了山fer酚，异鼠李素和刺五加素，但在Tx430中未发现这些黄酮醇（图1e）。值得一提的是，Tx430对照品中产生黄酮醇的小穗中没有注射器素（3'，5'- O-二甲基化黄酮醇）。注射器素的形成需要类黄酮3'，5'-羟化酶（F3'5'H）活性以生成3'，5'-羟化基团进行O-甲基化。在一些双子叶植物，属于P450 CYP75A F3'5'Hs亚科负责不同的类黄酮基板的5'-羟基化（Seitz的等人。，2006）。有趣的是，一种属于CYP75B亚家族的胆甾醇特异性5'-羟化酶参与了Tricin（3'，5'- O草中的二甲基化黄酮）生物合成（Lam et al。，2019）。在转基因SbF3H1品系的叶片组织中进行穿刺生物素合成需要CYP75A F3'5'H或CYP75B黄酮醇特异性5'-羟化酶。

黄酮醇合酶（FLS）是黄酮醇生物合成过程中F3H的下游酶。具有功能性特征的第一个单子叶植物FLS编码基因是玉米ZmFLS1，具有串联复制的基因ZmFLS2（Falcone Ferreyra等人，2012）。这两个基因在大多数玉米组织中组成性表达，并且可以通过UV-B处理进一步诱导。高粱SbFLS1是ZmFLS的高度保守同源物（> 96％的序列同一性）。同样，SbFLS1在Tx430对照中检查的所有组织中均表达（图1b），表明SbF3H1的表达可能调节黄酮的产生。因此，在小穗中检出了黄酮醇衍生物，但在缺乏SbF3H1的其他组织中未检出 在野生型高粱中表达。

黄酮衍生物是草类生物质中最常见的类黄酮代谢产物。一致的是，在小穗样品中检出芹菜素，金鸡油酚和甘油三酯，而在OE系中芹菜素和甘油三酯分别略有增加和减少（图1d）。同时，在旗叶样品中发现了芹菜素，木犀草素，金葱酚和三嗪。与Tx430对照相比，OE3中的芹菜素水平降低（图1e）。

高粱麸皮中含有大量的3-脱氧花青素（Awika et al。，2004）。一致地，在Tx430对照全种子提取物中检测到芹菜素和木犀草素（图1f）。有趣的是，在两种转基因品系的种子中都检测到了除黄酮醇外的花色素苷（pelargonidin和cyanidin），它们是3-羟基化的黄酮类化合物。花色素和3-脱氧花色素在结构上相似，除了C3-羟基化。由于种子中的内源性3-脱氧花青素分支途径是活跃的，因此某些酶可能接受3-羟基化的底物来进行花青素的生物合成。例如，两种二氢黄酮醇还原酶（SbDFR1和SbDFR3）可以还原二氢黄酮醇和黄烷酮，以分别生成花色素苷和3-脱氧花色素苷的直接前体（Liu等人，2010）。

拉长的中胚轴系统被进一步用于研究亚线虫感染后高粱幼苗中的类黄酮概况和疾病表型。LC-MS / MS分析显示，黄酮醇衍生物和花青素在已接种转基因幼苗的中胚轴中积累，但在Tx430中未积累（图1g）。同时，与Tx430幼苗相比，转基因幼苗减少了3-脱氧类黄酮植物抗毒素（3-脱氧花青素和黄酮）的积累。尽管感染后类黄酮的生物合成被大量激活（主要是通过查尔酮合酶的诱导），但SbF3H1显然使一些碳流转向了3-羟基化类黄酮的产生。

黄酮生物合成基因[黄酮合酶（SbFNSII； Sb02g000220），芹菜素3′-羟化酶/鞘脂5′-羟化酶（SbA3AH / C5ʹH； Sb09g022480）和类黄酮O-甲基转移酶（SbFOMT； Sb07g）的表达被检出中胚轴。在Tx430和两个转基因品系之间均未发现基因表达水平的显着变化，只是在OE3的旗叶中检测到SbFOMT的表达略有增加（数据未显示）。同时，表达3-脱氧花青素生物合成基因[黄烷酮4-还原酶（SbFNR； Sb06g029550）和SbDFR3（Sb04g004290）]在OE系的受感染中胚轴中未发生改变。这些结果表明，过表达SbF3H1不会导致其他类黄酮分支途径的基因表达发生重大变化。

对于接种的中胚轴，如所述（Schnippenkoetter et al。，2017），在接种后6天检查疾病的严重程度。与两个转基因中胚轴相比，Tx430中胚轴的强烈症状发生率高64％（图1h，i）。相比之下，没有疾病症状的转基因幼苗比Tx430对照多。因此，SbF3H1过表达导致对C的敏感性降低。亚线尽管产生的3-脱氧类黄酮植物抗毒素的数量较少。由于高粱病原体在野生型高粱的营养组织中较少遇到黄酮醇和花青素衍生物，因此真菌中可能不存在解毒机制。此外，这些3-羟基化的类黄酮可能与3-脱氧类黄酮类植物抗毒素协同作用，以提高过表达SbF3H1的植物的总体抗真菌能力。

高粱中胚轴中的过氧化氢和超氧离子分别通过二氨基联苯胺（DAB）和硝基蓝四唑（NBT）染色进行了分析（图1j）。在未接种的幼苗中，几乎没有DAB和NBT染色。接种后24小时，在Tx430对照中胚轴上有明显的褐色DAB损伤，而在转基因中胚轴上几乎观察不到。同样，与被感染的转基因幼苗相比，被感染的Tx430幼苗的NBT染色更为强烈和广泛。总体而言，在SbF3H1过表达株系中病原体诱导的ROS产生得以缓解。由于黄酮醇衍生物是有效的抗氧化剂（Heim等，2002），它们可以清除被感染的转基因幼苗中的一些ROS。

以前，转基因F3H过表达是在烟草中进行的，由于内源性F3H活性，烟草会自然积累3-羟基化的类黄酮，从而导致黄酮醇和黄烷-3-醇的积累增加，而类黄酮的分布没有剧烈变化（Song等人，2016）。相比之下，我们过表达SbF3H1在营养组织中缺乏3-羟基化黄酮的高粱中，导致部分碳流重新导向3-羟基化黄酮的产生，因此增加了不同组织中黄酮衍生的代谢物的复杂性。丰富的类黄酮分布可增强防御反应并改善高粱谷物/麸皮的营养价值。尤其是，以前未在高粱中鉴定出的新型黄酮类代谢物-注射素衍生物，具有一系列有益健康的特性（Hsu等，2009）。考虑到过表达品系的种子产量下降，应探索使用病原体诱导型和组织特异性启动子来驱动靶向SbF3H1 在不影响谷物生产率的前提下表达。