In our previous research, we firstly demonstrated blocking the light to the juvenile spike directly and qualitatively affected floret fertility by affecting anther greening and inducing pollen sterility in wheat. However, the underlying mechanisms remain unclear. Here, we integrated morphology, cytology, physiology and transcriptome to investigate the differences in spike fertility under control (CT) and light blocking (LB) treatments from jointing to either anthesis (LB_J-A) or booting (LB_J-B). Our results revealed that LB affected anther greening by disturbing chloroplast development and chlorophyll synthesis, suggesting that light received by the juvenile spike is essential for anther greening. The downregulation of genes involved in photosynthesis and chlorophyll biosynthesis suppressed the expression of enzymes in fatty acid, lignin, and starch metabolism, and combined with tapetum degradation resulted in abnormal pollen exine formation and pollen sterility. Collectively, this study elucidates the possible mechanism by which light blocking inhibits anther greening and leads to pollen sterility. These findings provide novel insights into light blocking regulating spike fertility, advancing the understanding of the regulation mechanism of reproductive development in wheat.

Light intensity plays a critical role in determining spike fertility of wheat (Triticum aestivum L.) by influencing floret development. However, whether a light signal directly perceived by the juvenile spike is essential for spike fertility remains unknown. To address this, we conducted two experiments imposing light blocking (LB) treatments from jointing to either anthesis (LB_J-A) or booting (LB_J-B) as well as a control (CT). LB was imposed through an innovative procedure employing aluminum foil. We found that LB_J-A did not affect floret primordia initiation, only increased distal floret mortality, and drastically impaired pollen viability in the anthers, resulting in a slight reduction in the number of competent florets but a massive sterility of all florets across all spikelets compared to CT, and LB_J-B exhibited results closer to the CT. For instance, under field conditions, the number of fertile florets was 42.92 in CT and 36.10 in LB_J-B, whereas no fertile florets (0) were observed in LB_J-A. While re-exposing juvenile spikes to light at booting restored fertility, highlighting that light signal perceived by juvenile spike from booting to anthesis played a crucial role for floret fertility. Furthermore, a strong correlation was observed between the number of fertile florets and the number of floret primordia with green anthers, suggesting that light blocking may impair fertility by preventing anther greening. These novel findings provide evidence that light signal directly perceived by juvenile spikes qualitatively may affect spike fertility possibly through affecting anther greening and inducing sterile pollen formation.

Promoting more floret primordia within a spike to acquire fertile potential during the differentiation and pre-dimorphism phases is critical for increasing the number of fertile florets per spike (NFFs). However, it is yet unknown the physiological mechanism regulating the complex and dynamic process. This study aimed to clarify how intra-spike hormones, pigments, and assimilates coordinate with each other to regulate spike morphology and then floret primordia development. A two-year field experiment was conducted with two winter wheat genotypes: N50 (big-spike with greater NFFs) and SM22 (medium-spike with fewer NFFs). We monitored high temporal and spatial-resolution changes in the number and morphology of floret primordia within a spike, as well as in intra-spike hormones, pigments, and assimilates. Our results revealed that the big-spike genotype had more NFFs than the medium-spike genotype, not only because they had more spikelets, but also because they had greater NFFs mainly at central spikelets. More floret primordia at central spikelets had sufficient time to develop and acquire fertile potential during the differentiation phase (167–176 d after sowing, DAS) and the pre-dimorphism phase (179 DAS) for the big-spike genotype than the medium-spike genotype. Floret primordia with fertile morphology during the pre-dimorphism phase always developed into fertile florets during the dimorphism phase. Those early-developed floret primordia most proximal and intermediate to the rachis in the big-spike genotype developed faster than the medium-spike genotype. Correspondingly, the spike dry matter and pigments (chlorophyll a, chlorophyll b, carotene, and carotenoids) content during 170–182 DAS, auxin (IAA) and cytokinin (CTK) content on 167 DAS were significantly higher in the big-spike genotype than in the medium-spike genotype, while jasmonic acid (JA) content was significantly lower in the big-spike genotype compared to the medium-spike genotype during 167–182 DAS. Since the significant differences in intra-spike hormone content of the two genotypes appear earlier than those in dry matter and pigments, we propose a possible model that helped the N50 genotype (big-spike) to form more fertile florets, taking the intra-spike hormone content as a signaling molecule induced assimilates and pigments synthesis, which accelerated the development of more floret primordia during the differentiation phase and then acquired fertile potential during the pre-dimorphism phase, finally improved the NFFs. Our high temporal and spatial-resolution analysis provides an accurate time window for precision cultivation and effective physiological breeding to improve the number of fertile florets in wheat.

Nitrogen (N) fertilization is critical for spike and floret development, which affects the number of fertile florets per spike (NFFs). However, the physiological regulation of the floret development process by N fertilization is largely unknown. A high temporal-resolution investigation of floret primordia number and morphology, dry matter, and N availability was conducted under three N fertilization levels: 0 (N0), 120 (N1) and 240 (N2) kg ha⁻¹. Interestingly, fertile florets at anthesis stage were determined by those floret primordia with meiotic ability at booting stage: meiotic ability was a threshold that predicted whether a floret primordium became fertile or abortive florets. Because the developmental rate of the 4th floret primordium in the central spikelet was accelerated and then they acquired meiotic ability, the NFFs increased gradually as N application increased, but the increase range decreased under N2. There were no differences in spike N concentration among treatments, but leaf N concentration was increased in the N1 and N2 treatments. Correspondingly, dry matter accumulation and N content of the leaf and spike in the N1 and N2 treatments was increased as compared to N0. Clearly, optimal N fertilization increased leaf N availability and transport of assimilates to spikes, and allowed more floret primordia to acquire meiotic ability and become fertile florets, finally increasing NFFs. There was no difference in leaf N concentration between N1 and N2 treatment, whereas soil N concentration at 0–60 cm soil layers was higher in N2 than in N1 treatment, implying that there was still some N fertilization that remained unused. Therefore, improving the leaf’s ability to further use N fertilizer is vital for greater NFFs.

Increased grain yield (GY) and grain protein concentration (GPC) are the two main targets of efforts to improve wheat (Triticum aestivum L.) production in the North China Plain (NCP). We conducted a three-year field experiment in the 2014–2017 winter wheat growing seasons to compare the effects of conventional irrigation practice (CI) and micro-sprinkling irrigation combined with nitrogen (N) fertilizer (MSI) on GY, GPC, and protein yield (PY). Across the three years, GY, GPC, and PY increased by 10.5%–16.7%, 5.4%–8.0%, and 18.8%–24.6%, respectively, under MSI relative to CI. The higher GY under MSI was due primarily to increased thousand-kernel weight (TKW). The chlorophyll content of leaves was higher under MSI during the mid–late grain filling period, increasing the contribution of post-anthesis dry matter accumulation to GY, with consequent increases in total dry matter accumulation and harvest index compared to CI. During the mid–late grain filling period, the canopy temperature was markedly lower and the relative humidity was higher under MSI than under CI. The duration and rate of filling during the mid–late grain filling period were also higher under MSI than CI, resulting in higher TKW. MSI increased the contribution of post-anthesis N accumulation to grain N but reduced the pre-anthesis remobilization of N in leaves, the primary site of photosynthetic activity, possibly helping maintain photosynthate production in leaves during grain filling. Total N at maturity was higher under MSI than CI, although there was little difference in N harvest index. The higher GPC under MSI than under CI was due to a larger increase in grain N accumulation than in GY. Overall, MSI simultaneously increased both GY and GPC in winter wheat grown in the NCP.