Introduction

A suitable temperature is a necessary condition for the survival of organisms, but the adaptability of various organisms to changes in temperature differs considerably1. A significant difference between plants and other multicellular complex organisms is that plants live in a fixed state. Therefore, throughout their life cycle, plants are forced to endure a variety of adverse environmental conditions, among which low temperature is one of the main limiting factors affecting the natural distribution and crop yield of plants2. In subtropical to frigid regions, overwintering plants have evolved strategies to adapt to the low-temperature environment. Deciduous fruit trees need to experience a certain period of low temperature to undergo natural dormancy to normally blossom and bear fruit3. However, too low of a temperature will lead to the freezing of branches, reductions in fruit yield and fruit quality, and even death4.

After a chilling injury occurs, plant hormone levels are adjusted to reduce the impact on growth and development. As an important hormone regulating anther development, gibberellic acid (GA) plays an important role in resisting chilling injury. Previous studies have shown that bioactive GAs (GA1, GA3, GA4, and GA7) function in pollen exine formation and programmed cell death of tapetal cells in Arabidopsis5, rice6, tomato7, and peach8. In addition, cold treatment damages anthers in the young microspore stage and induces severe pollen sterility and yield reduction, which can be partially recovered by spraying with GA9. However, how and why GA functions in almond reproductive processes under normal and low temperatures remain unclear. Previous research has shown that the B3 domain-containing transcription factor FUS3 plays a role in controlling the GA-ABA balance10 and that GA and abscisic acid (ABA) act antagonistically to control Arabidopsis development. ABA-induced by low temperature is involved in the degradation of the tapetum11, and the accumulation of ABA affects sugar transport, resulting in rice pollen sterility12. In addition, GA interacts with other hormones, including jasmonic acid (JA)13, indole-3-acetic acid (IAA)14, cytokinins15, and salicylic acid (SA)16, to participate in plant development. JA promotes the growth of stems and anthers through MYB genes17, and 24 GA-responsive genes and 82 JA-responsive genes work together to regulate the development of rice anthers18. IAA is required for the development of diploid microsporocyte cells, and IAA affects GA1 biosynthesis by maintaining PsGA3ox1 gene transcript levels19; furthermore, GA promotes IAA accumulation through the GA-DELLA-LEC1 (LEAFY COTYLEDON 1) signaling cascade20. In the rice (Oryza sativa) meristem, the KNOTTED1-like homeobox (KNOX) protein activates cytokinin biosynthesis by upregulating the isopentenyl transferase (IPT) gene and subsequently reduces the expression of GA2ox20. In tomato, SA, GA, and antioxidant enzymes can interact with one another and protect tomato fruit from chilling injury and oxidative damage21.

Almond (Amygdalus communis L.) is considered the most important tree nut crop worldwide22. However, its nut yield is approximately 500 kg per hectare in Xinjiang, China, which is only one-quarter of that in California, USA. The low yield of almonds is mainly caused by the extremely low-temperature climate in the winter and early spring. In China and other Eurasian countries, almond trees require chilling to develop fruiting buds21. From autumn to winter, a reduction in gibberellin content under short photoperiod and chilling temperatures affects almond bud development and dormancy, and reproductive tissues become resistant to freezing23. However, extremely low temperatures below −20 °C occurring in the winter can damage reproductive tissues, including by having effects on microsporogenesis, which is susceptible to low temperatures, leading to severe reductions in nut yields24. Under increasing gibberellin levels and temperatures, anther reproductive tissue develops quickly and becomes less resistant to frost25. To increase the almond yield and reduce the losses caused by freezing damage, a number of experiments have been performed and have shown that different almond varieties exhibit differences in resistance26. In response to low temperature, the expression of GA-related genes is altered in almond anthers4; in the regulation of almond anthesis, C-repeat binding factor (CBF) and gibberellin 20 oxidase (GA20OX), which are involved in gibberellin biosynthesis, work together to regulate flower bud break. Spraying with GA3 either alone or in combination with potassium nitrate (KNO3) or benzyl adenine (BA) during the winter can induce earlier bud burst and increase the percentage of floral budburst, fruit set, and branch induction27.

The Chinese almond industry is limited by cold winds from Siberia, and the breeding of new almond varieties with high quality, high yields, and cold resistance is the only way to further develop the almond industry in Xinjiang. Extremely low temperatures reduce bioactive GA levels and induce severe pollen sterility, which can be restored by exogenous GA3. As a woody plant, the almond has evolved many specific strategies to respond to low temperatures. Whether GA functions in the mediation of almond anther development under cold conditions and how GA interacts with other hormones during anther development remain unknown. In this study, we analyzed the phenotypic and physiological changes in almond anthers and explored GA-regulated networks during anther development using RNA-seq and qRT-PCR. We obtained the novel finding that GA interacts with other hormones to control anther development and screened out key genes related to high-quality resistant pollen that may be used to breed new almond varieties.

Results

Anther development is associated with phytohormone levels

To explore the relationships between anther development and the levels of endogenous GAs, we first analyzed microspore development in almond. The results showed that the growth rate per 1000 anthers was 0.10 mg/d in ZP1 (“Zhipi” almond at the meiosis stage in the field), 0.21 mg/d in ZP2 (“Zhipi” at the tetrad stage in the field), and 3.38 mg/d in ZP3 (“Zhipi” at the 1-nucleus stage in the field) and then fell to 0.69 mg/d in ZP4 (“Zhipi” at the mature 2-nucleus stage in the field) (Table 1), indicating that rapid growth of anthers occurs from the tetrad stage to the 1-nucleus stage.

Table 1 Weight and growth rate per 1000 anthers
Full size table

The ZP1–ZP3 anthers exhibited five cell layers, whereas the ZP4 anthers presented three cell layers. The middle-layer cells were small and exhibited small vacuoles in the ZP1 anthers, began to undergo degradation in the ZP2 anthers, and were almost completely degraded in the ZP4 anthers. The multinucleate secretory tapetum exhibited a clear boundary and small vacuoles in the ZP1 anthers; the vacuoles in this structure became larger in the ZP2 and ZP3 anthers, and the multinucleate secretory tapetum had completely disappeared in the ZP4 anthers. The meiotic cells in the ZP1 anthers contained many fat droplets, starch granules, and small vacuoles, and some meiotic cells contained replicating DNA. The tetrad cells in the ZP2 anthers contained larger vacuoles, were covered by sporopollenin, and showed close positioning of the nucleus to the pollen wall. The 1-nucleus pollen in the ZP3 anthers presented a small number of starch granules and fat droplets and was covered by sporopollenin. Finally, the mature 2-nucleus pollen of the ZP4 anthers contained more fat droplets and starch granules but no vacuoles and was covered by sporopollenin (Fig. 1).

Fig. 1: Analyses of anther development in almond.
figure 1

ZP1, meiosis stage in the field; ZP2, tetrad stage in the field; ZP3, 1-nucleus stage in the field; and ZP4, mature 2-nucleus stage in the field. Anthers were photographed using a camera; the sections were photographed using microscopes; the anther wall and pollen cells were observed using SEM, and the pollen nucleus was stained with DAPI and photographed under a microscope

Full size image

GA and other hormones function together in anther development in almonds. We sampled the anthers at four developmental stages to test the levels of hormones using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS). The results indicated that the levels of GA4, which mainly functions in anther development, increased significantly with pollen development. GA7 was detected at higher levels in the ZP1 and ZP2 anthers. Accordingly, their precursors, namely, GA24 and GA9, were present at relatively low levels. Other bioactive GAs, such as GA1 and GA3, were detected in only the ZP3 anthers, whereas their precursors, including GA44 and GA20, showed high levels. These results indicated that bioactive GAs mainly functioned at the 1-nucleus pollen stage and that the growth rate per 1,000 anthers exhibited a significant positive correlation with the total bioactive GA compound content (Table 2). We also examined the levels of other hormones, including ABA, JA, trans-zeatin (tZ), IAA, and SA. The levels of ABA, the antagonistic hormone of GA, decreased from ZP1 to ZP3 but rapidly increased in ZP4 anthers. However, the levels of JA, tZ, IAA, and SA were much higher in the ZP3 anthers and exhibited a significant decline in the ZP4 anthers (Table 2). These results suggested that GA might exert an effect similar to that of JA, tZ, IAA, and SA and opposite to that of ABA in regulating almond anther development.

Table 2 Plant hormone levels (ng/g FW)
Full size table

GA affects anther development and the levels of other hormones in anthers

To understand the function of GA in almond anther development, we treated almond flowers with bioactive GA, GA3, and the GA biosynthesis inhibitor paclobutrazol (PP333). The results indicated that the growth rate per 1000 anthers in the water control group (CK) sample was 2.16 mg/d, which was higher than that in the PP333-treated sample (1.88 mg/d) and lower than that in the GA3-treated sample (2.74 mg/d) (Table 1). The sectioning results showed that these anthers were all in the 2-nucleus stage and exhibited 4 types of cells, including epidermal cells, endothelial cells, middle-layer cells, and tapetal cells. To further explore the differences among these three treatments, we used scanning electron microscopy to observe the anther microstructure. We found that epidermal cells, endothelial cells, and middle-layer cells in PP333-treated anthers were thinner than those in CK anthers, whereas these layers were the thickest in GA3-treated anthers. The tapetal cells were secretory and began to be degraded in CK anthers; they were degraded faster in GA3-treated anthers and more slowly in PP333-treated anthers. Furthermore, the microspores contained more fat droplets and starch granules and exhibited a richer sporopollenin cover in GA3-treated anthers than in the other two sample groups. The cytoplasm and nucleus of tapetal cells in PP333-treated anthers showed less degradation than those in CK and GA3-treated anthers. The pollen from PP333-treated anthers exhibited two nuclei, the largest vacuoles, small numbers of fat droplets and starch granules, and little sporopollenin cover. The pollen from CK anthers exhibited two nuclei, large vacuoles, some fat droplets and starch granules, and some sporopollenin cover. The pollen from GA3-treated anthers exhibited two nuclei, small vacuoles, many fat droplets and starch granules, and greater sporopollenin cover (Fig. 2).

Fig. 2: Function of GA in pollen development.
figure 2

A total of 600 branches from three trees at the ZP3 stage (1-nucleus stage) were treated at −12 °C until the relative sensitivity of conductivity in the buds reached approximately 50%, after which they were placed in a 4 °C climatic cabinet for 24 h. The treated samples were equally divided into three groups: a 100 μmol/L GA3-treated group and a CK group and a 100 μmol/L PP333-treated group. The branches were fixed in flower putty containing 1/2 MS+5% sucrose hydroponics in a climatic cabinet for 3 days. The other branches were cultivated to bloom in a climatic cabinet; their mature anthers were collected to test pollen viability. The anthers were photographed using a camera; the sections were photographed using microscopes; the anther wall and pollen cells were observed using SEM; the pollen nuclei were stained with DAPI and photographed under a microscope, and pollen viability was observed by microscopy

Full size image

To determine whether GA affected pollen viability, we sampled mature pollen from samples subjected to the three treatments to perform a germination assay. In the PP333-treated anthers, 42.00% of the pollen germinated, which was significantly lower than the percentage in CK anthers (49.33%); the pollen germination rate was also higher in the GA3-treated anthers (67.66%). Moreover, the number of pollen grains per anther was 1465 in the PP333-treated anthers, 1624 in the CK anthers, and 1804 in the GA3-treated anthers (Table 3). The results suggested that GA plays a role in increasing pollen quantity and vigor in almonds.

Table 3 Pollen quantity per anther and pollen viability
Full size table

In the hormone assays, we found that the levels of IAA, tZ, and JA were higher in GA3-treated anthers than in CK anthers, whereas there was no significant difference between PP333-treated and CK anthers. In contrast, the levels of ABA and SA were decreased in the GA3-treated group but exhibited no significant difference between the PP333-treated and CK groups (Table 2). These results regarding hormone levels combined with the phenotypes of anther development suggested that GA may regulate anther development by mediating the levels of different hormones.

Analyses of transcriptome dynamics and module-trait relationships

To study unigene dynamics and identify the candidate genes in anthers affected by exogenous GA3 and its inhibitor PP333, mRNA from four field samples and three treated samples at ZP3 (1-nucleus pollen stage) was sequenced. A total of 21,024 genes were detected in the 4 field samples, 13,686 of which were coexpressed (Fig. 3a), and 3030 differentially expressed genes (DEGs) were identified among them (Fig. 3b).

Fig. 3: Differentially expressed unigenes in anthers and the module–trait relationships among genes, phytohormone levels, and growth rate per 1000 anthers.
figure 3

a Venn diagram of differentially expressed genes in the anthers of the four field samples. b Venn diagram of coexpressed genes in the anthers of the four field samples. c Venn diagram of differentially expressed genes in the anthers of the three treated samples. d Venn diagram of coexpressed genes in the anthers of the three treated samples. e Module-trait relationships among the genes, phytohormone levels, and growth rate per 1000 anthers

Full size image

In the 3 treated samples, a total of 16,848 genes, including 16,172 coexpressed genes, were detected (Fig. 3c). Among these genes, 89 genes were regulated by exogenous GA3, and 91 genes were affected by PP333 (Fig. 3d). To explore the key genes involved in GA-regulated anther development in almond, we analyzed the module-trait relationships among the gene fragments per kilobase million (FPKM) values, phytohormone levels, and growth rate per 1000 anthers. The results indicated that the growth rate per 1,000 anthers exhibited a significant positive correlation with the gene FPKM values in modules 1, 27, and 29 but a significant negative correlation with the gene FPKM values in modules 17 and 22 at the 0.05 level (Fig. 3e).

Analysis of reprogramming genes in anthers

In GA biosynthesis and signal transduction, GA12 is the first plant gibberellin converted from trans-geranylgeranyl diphosphate (GGDP) by the ent-copalyl diphosphate synthase/GA requiring 1 (CPS/GA1), ent-kaur-16-ene synthase/GA requiring 2 (KS/GA2), ent-kaurene oxidase/GA requiring 3 (KO/GA3), and ent-kaurenoic acid oxidase (KAO) enzymes14. The gibberellin 20 oxidase (GA20OX) and gibberellin 3-beta-dioxygenase (GA3OX) enzymes promote the conversion of GA12 to bioactive GAs, and gibberellin 2-beta-dioxygenase (GA2OX) enzymes catalyze the 2-beta-hydroxylation of bioactive GA28. In the cytoplasm, the GA-GID1-SLR1 complex can be degraded through the participation of Gibberellin-Insensitive Dwarf 2 (GID2), leading to changes in transcription factors (TFs) and GA-responsive gene expression29. In this experiment, the content of total bioactive GA compounds showed a significant positive correlation with the gene FPKM values in modules 9, 14, 27, and 28 and a significant negative correlation with the gene FPKM values in module 17 (Fig. 3e). GA20OX2 in module 27 and GID2 in module 2 were downregulated, while the DELLA protein gene gibberellin-insensitive (GAI) in module 27 was upregulated, after treatment with exogenous GA3; GA2OX2 in module 2 was downregulated by PP333. There were 20 TFs that were affected by exogenous GA3. Dehydration-responsive element-binding protein 1A (DREB1A), basic helix–loop–helix transcription factors (bHLH35, bHLH83, bHLH92, BIM1, HEC1), WRKY transcription factors (WRKY33, WRKY41, and WRKY53), MYB transcription factor 35 (MYB35), trihelix, MADS-box transcription factor 23 (MADS23), and common plant regulatory factor 1 (CPRF1) were upregulated by exogenous GA3, while Reveille 1 (RVE1) was downregulated by exogenous GA3. Module-trait relationships showed that the FPKM values of MYB4, MYB44, bHLH35, organ weight 2 (bHLH38/ORG2), bHLH92, HEC1, WRKY33, WRKY41, and WRKY53 were obviously positively correlated with the growth rate per 1000 anthers, while the FPKM value of RVE1 was obviously negatively correlated with the growth rate per 1000 anthers. An additional 84 genes were affected by either exogenous GA3 or PP333 (Fig. 4a).

Fig. 4: Heatmap of representative genes in six phytohormone pathways.
figure 4

a GA biosynthesis and signal transduction; b ABA biosynthesis and signal transduction; c tZ biosynthesis and signal transduction; d JA biosynthesis and signal transduction; e IAA biosynthesis and signal transduction; and f SA biosynthesis and signal transduction. Genes with red circles showed significant changes after GA3 spraying, while genes with blue squares showed significant changes after PP333 spraying. The module number is given after the gene name and gene ID

Full size image

ABA is converted from zeaxanthin and deactivated by ABA 8′-hydroxylase (ABAH)14. In the ABA signaling pathway, ABA is perceived by the ABA receptor pyrabactin resistance 1 (PYR1)/PYR1-like (PYL) proteins, which then deliver the signal to phosphatase 2C (PP2C) proteins30. PP2C proteins are negative regulators of the ABA pathway that prevent sucrose nonfermenting 1 (SNF1)-related protein kinase 2 (SnRK2) from activating downstream signals, such as ABA-responsive element-binding factor31. In this experiment, the ABA content showed a significant positive correlation with the gene FPKM values in modules 1, 3, 5, 6, and 16 and a significant negative correlation with the gene FPKM values in modules 2, 10, and 29 (Fig. 3e). Short-chain dehydrogenase reductase 1/ABA-deficient 2 (SDR1/ABA2) in module 3 was downregulated by exogenous GA3; ABAH1 and phosphatase 2C 25 (P2C25) in module 27 were upregulated by exogenous GA3, and abscisic acid-insensitive 5 (ABI5) in module 27 was downregulated by PP333 (Fig. 4b).

In tZ biosynthesis and signal transduction, IPT and cytokinin hydroxylase (C735A) promote the synthesis of trans-zeatin riboside monophosphate (tZRMP) from dimethyl diphosphate (DMAPP) together with ATP, ADP, and AMP32. tZRMP is converted to tZ with the participation of phosphatase and glycosidase, tZ is in turn deactivated by cytokinin oxidase (CKX), and a UDP-glycosyltransferase (UGT) superfamily protein bound to a soluble receptor histidine kinase (HK) transfers a phosphate group to a histidine-containing phosphotransferase (HP) protein and a two-component response regulator ARR protein to regulate cell division14. In this experiment, the tZ content showed a significant positive correlation with the gene FPKM values in modules 1, 14, 20, and 27 and a significant negative correlation with the gene FPKM values in module 17 (Fig. 3e). AHP6 and ARR9 in module 2 and ARR17 in module 10 were downregulated by exogenous GA3 (Fig. 4c).

In JA biosynthesis and signal transduction, JA synthesis from lecithin is carried out by seven enzymes, with lipoxygenase (LOX) in the chloroplast being the rate-limiting enzyme in this process14. JA can be transformed to methyl jasmonate (MeJA) via the action of jasmonate O-methyltransferase (JMT) and to jasmonoyl-l-isoleucine (JA-Ile) via the action of JA-amido synthetase (JAR1)33. JA-Ile can disrupt TIFY domain/jasmonate-zim-domain (TIFY/JAZ) proteins together with a coronatine-insensitive (COI) protein and MYC2 to regulate JA-responsive genes34. In this experiment, the JA content showed a significant positive correlation with the gene FPKM values in modules 27 and 29 and a significant negative correlation with the gene FPKM values in modules 3 and 17 (Fig. 3e). LOX31 in module 29, TIFY9 in module 3, and TIFY10A in module 27 were upregulated by exogenous GA3, whereas JMT in module 3 was downregulated by exogenous GA3. TIFY5A in module 1, TIFY9 in module 3, and TIFY10A in module 27 were upregulated by PP333 (Fig. 4d).

In IAA biosynthesis and signal transduction, IAA is synthesized from tryptophan (Trp) in two parallel pathways and can release free IAA conjugates under the action of IAA-amino acid hydrolase ILR1-like 1 (ILL1)35. IAA is transported by auxin-responsive protein 1 (AUX1) and auxin transporter-like (LAX) proteins36 and is perceived by transport inhibitor response (TIR) proteins37, which can regulate the expression of IAA-responsive genes, including the auxin response factor (ARF), AUX/IAA, indole-3-acetic acid-amido synthetase GH3 and SMALL AUXIN UPREGULATED RNA (SAUR) genes, to further influence cell enlargement and plant growth14. In this experiment, the IAA content was significantly positively correlated with the gene FPKM values in modules 27 and 29 and significantly negatively correlated with the gene FPKM values in modules 3 and 17 (Fig. 3e). ILL1 in module 10 was upregulated by exogenous GA3 and PP333, whereas indole-3-acetic acid-amido synthetase GH3.1 (GH3.1) in module 27 and indole-3-acetic acid-induced protein ARG7 (ARG7) in module 2 were downregulated by PP333 (Fig. 4e).

In SA biosynthesis and signal transduction, SA is synthesized from chorismate via two parallel pathways, in which the isochorismate synthase (ICS) pathway might be the main pathway38. There are several ways to reduce SA levels. UDP-glycosyltransferase 74F (U74F/SAG) transfers glucose to SA, forming a glucoside (SAG)39, and salicylate carboxymethyltransferase (SAMT) catalyzes the biosynthesis of methyl SA, which can be converted by SA-binding protein (SABP). Nonexpressor of pr genes 1 (NPR1) is the key regulator of SA-JA crosstalk, mediates the binding of bZIP transcription factor family (TGA) members to the pathogenesis-related basic form of pathogenesis-related protein 1 (PRB1), and induces systemic acquired resistance (SAR) and defenses against abiotic stresses and disease40. In this experiment, the SA content exhibited a significant positive correlation with the gene FPKM values in modules 1, 20, and 27 and a significant negative correlation with the gene FPKM values in modules 3 and 17 (Fig. 3e). Only SABP2 in module 10 was downregulated by exogenous GA3 (Fig. 4f).

Validation of RNA-seq data

To evaluate the concordance of the gene expression intensities between RNA-seq and qRT-PCR, 20 unigenes related to GA3 treatment or PP333 treatment were selected to perform qRT-PCR to confirm the RNA-Seq data; moreover, the correlations of the changes in 20 unigenes between them were also investigated. High expression correlations, with high correlation coefficients (R2 > 0.75), were observed between the RNA-seq and qRT-PCR data (Fig. 5, Table S2). Most importantly, the expression levels of these selected genes determined by qRT-PCR were basically consistent with the RNA-seq results (Fig. 5, Table S2).

Fig. 5: Validation of the expression levels of 20 unigenes.
figure 5

Relative expression levels of 20 hormone-related genes were tested by qRT-PCR method to confirm the RNA-seq data. The samples include field samples (ZP1, ZP2, ZP3, and ZP4) and treated samples (PP333 -treated, GA3 -treated, and CK). The values represent the means ± SD of three biological replicates

Full size image

Regulatory network of GA affecting anther development

To elucidate the regulatory network of GA in the regulation of anther development, all DEGs related to the biosynthesis and signal transduction of six phytohormone pathways were selected based on the module–trait relationships of the gene FPKM values, phytohormone levels, and growth rate per 1000 anthers. The regulatory network was constructed by referring to the known protein–protein interactions in Arabidopsis. The regulatory network showed that GA regulated almond anther development by interacting with five phytohormone pathways and the DREB1A-mediated signaling pathway, which included the phytohormone biosynthesis and signal transduction genes GA20ox2, GID2, GAI, SDR1, ABAH1, P2C25, ABI5, ARR5, ARR9, ARR17, AHK1, AHP6, ILL1, CH3.1, ARG7, LOX3, JMT, ADH1, TIFY5, TIFY9, TIFY10A, DIOX2, SABP2, and 1-aminocyclopropane-1-carboxylate synthase 1 (1A11/ACS); the transcription factor genes DREB1A, MYB35, MYB44, RVE1, bHLH35, HEC1, and WRKY33; and the GA-responsive genes oligopeptide transporter 3 (OPT3), zinc transporter 4 (ZIP4), ABC9 transporter G family member 26 (ABCG26), 4-coumarate-CoA ligase-like 1 (4CLL1), protein NRT1/PTR FAMILY 5.14 (PTR9), senescence-associated gene 13 (SAG13), kinase 2 (KIN2), and responsive to desiccation 22 (RD22), among others (Fig. 6).

Fig. 6: Regulatory network of GA in the development of almond pollen.
figure 6

ACOS5 = 4CLL1; AEE14 = MENE; ALDH11A3 = GAPN; APG3 = CPRF1; At1g10970 = ZIP4; At1G20270 = P4H3; AT1G22540 = PTR9; At2g30020 = P2C25; AT5G38710 = PROD2; AT2G42940 = AHL16; At3g47570 = Y3475; At5g56590 = E1313; AT5G57150 = bHLH35; AT5G66120 = DHQSD; CASP3 = CSD; ChlAKR = AKR4C9; CYP707A1 = ABAH1; EDS16 = ICS1; MES16 = SABP2; ICL = ACEA; MYBR1 = MYB44; TDF1 = MYB35

Full size image

Discussion

GA is an important hormone involved in regulating anther development in plants41. A significantly reduced level of GA results in pollen sterility42. Moreover, GA plays an important role in cold responses during the reproductive stage in rice43. However, the function of GA in mediating pollen development in woody plants is less well studied. Therefore, the goal of this work was to determine whether and how GA functions in almond reproductive development. We analyzed microspore development and measured the hormone levels in the anthers at four pollen development stages. The results showed that the total bioactive GA compound levels gradually increased from ZP1 to ZP3 and that the correlation between the growth rate per 1000 anthers and the total bioactive GA compound content was 0.981, representing a significant correlation at the 0.05 level (two-sided). Our results indicate that bioactive GA compounds play important roles in almond anther development under normal- and low-temperature conditions.

GA interacts with other hormones in almond anthers

Herein, we found that exogenous GA3 increased the levels of IAA, tZ, and JA and decreased the levels of SA and ABA. The balance of hormones is critical for pollen development and low-temperature responses; the balance between pollen development and cold tolerance is controlled by hormone homeostasis. IAA plays a role in the pollen production ability and fertility of spikelets. The increased level of IAA in almond in the GA treatment group helped to improve pollen vigor and fertility under normal- and low-temperature conditions. Cytokinin regulates anther development by coordinating carbon and nitrogen metabolism. In this study, we found that tZ, a kind of cytokinin, was induced by GA in almond anthers, showing that GA can stimulate the biosynthesis of cytokinins to regulate anther development. JA also affects flower development in plants, showing a higher level during anther development. The level of JA induced by GA contributes to improving pollen fertility under cold conditions. GA and ABA are antagonistic hormones, so the ABA level was reduced in GA-treated almond anthers in this study. However, JA can stimulate ABA overproduction to upregulate plant abiotic stress tolerance. Taken together, the results of our hormone analysis suggest that hormone balance regulated by GA is important for correct anther development.

We next sought to determine how GA regulates other hormones. Our RNA-seq data showed that GA affected the expression of many hormone biosynthesis-related genes. For example, the expression of the ABA biosynthesis gene ABA2 was decreased, while the expression of the ABA-degradation gene ABAH1 was increased, by exogenous GA3, indicating that GA reduces ABA levels. Moreover, GA3 induced the expression of LOX3, a key enzyme gene in JA synthesis, thereby increasing JA levels and contributing to anther development in almond. Furthermore, we found that signal transduction genes presented altered expression in the GA-treated group. These results imply that GA interacts with other hormones to regulate the expression of genes involved in their biosynthesis and thereby affects related signaling pathways.

GA regulates the development of almond anthers

We found that exogenous GA3 promoted epidermal cell and endothecium cell elongation, tapetal cell degradation, and the accumulation of fat droplets and starch granules within pollen and sporopollenin on the pollen surface, while PP333 exerted the opposite effects. Exogenous GA3 increased the growth rate per 1000 anthers, pollen viability and the number of pollen grains per anther. To determine how GA regulated the development of almond anthers, we selected all the DEGs among the three treatment groups and constructed a regulatory network by referring to the known protein–protein interactions of Arabidopsis; we found that seven pathways might play a role in regulating the development of almond anthers (Fig. 6).

In the first pathway, the HEC1 protein showed an interaction with the AHP6 protein of the tZ pathway, and the FPKM value of HEC1 in module 27 was upregulated by exogenous GA3 and positively correlated with the growth rate per 1000 anthers. A previous study showed that HEC1 might act as a local modulator of IAA and tZ to control gynoecium development42. Therefore, we propose that HEC1 might be a local modulator of the GA and tZ pathways to control anther development.

In the second pathway, the FPKM value of bHLH38/ORG2 in module 27 was upregulated by PP333 and positively correlated with the growth rate per 1000 anthers, and the FPKM value of OPT3 outside of the 29 modules was upregulated by both exogenous GA3 and PP333. Previous evidence has shown that bHLH38/ORG2 plays a role in Fe regulation43 and that OPT3 is involved in Fe, Zn, Cd, and oligopeptide translocation44. Therefore, we surmise that bHLH38/ORG2 and OPT3 function in anther development as nutrient transporters.

Based on the third pathway, the expressions of ZIP4, 4CLL1/ACOS5, and prolyl 4-hydroxylase 3 (P4H3) were positively correlated with the growth rate per 1000 anthers. The expression of ZIP4 and 1A11/ACS was elevated by both GA3 and PP333; the levels of MYB35 and P4H3 were induced by exogenous GA3; and the expression of ABCG26 and 4CLL1/ACOS5 was repressed by PP333. Evidence shows that MYB35/TDF1 is located downstream of dysfunctional tapetum 1 (DYT1) in the control of tapetal development and pollen wall formation45. ZIP4 is considered an important Zn uptake transporter46; ABCG26 plays a role in pollen exine formation by exporting polyketide traffic47; 4CLL1/ACOS5, which is mainly expressed in the tapetum, is required for pollen wall exine formation48; and P4H3 catalyzes the formation of 4-hydroxyproline for the construction of plant cell wall glycoproteins49. Therefore, nutrient transport, regulation of pollen exine formation and tapetum degradation, and ethylene biosynthesis are critical for anther development in almond.

In this study, the expression of respiratory burst oxidase protein F (RBOHF) in module 2 and PTR9 in module 27 was increased by exogenous GA3 and was positively correlated with the growth rate per 1000 anthers. Previous studies have shown that RBOHF is induced by low temperature, drought, salt, ABA, and H2O2 and mediates diverse physiological processes50 and that PTR9 is involved in oligopeptide transport51. Thus, the available data indicate that GA regulates anther development by improving oligopeptide transport and stress responses.

Moreover, we found that WRKY53, SAG13, DHQSD (bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase), MENE/AAE14, and CPRF1/APG3 levels were elevated by GA and that aldo-keto reductase family 4 member C9 (AKR4C9) and glucan endo-1,3-beta-glucosidase (E1313) levels were reduced by GA. WRKY53 and SAG13 function in the regulation of senescence52. AAE14/MENE is involved in phylloquinone biosynthesis53; CPRF1/APG3 plays an important role in chloroplast development54, and DHQSD catalyzes the second step in the shikimate pathway55. E1313/At5g56590 is involved in the degradation of callose walls around the microspore tetrad and is essential for pollen exine formation56. Based on these data, we speculate that GA regulates anther tissue senescence by interacting with SA to affect anther development in almond.

GA regulates the resistance of anthers to low temperature

In our experiment, some stress-responsive genes, including DREB1A, bHLH92, WRKY41, KIN2, and ADH1, were induced in the GA-treated groups. Most of these genes showed obvious positive correlations with the growth rate per 1000 anthers. Previous studies have revealed that DREB1A plays a crucial role in cold acclimation and freezing resistance by positively regulating DELLAs and thereby inducing the expression of KIN2 and RD2257. ADH1 enhances the freezing resistance of plants46. bHLH92 functions in the osmotic stress response58. MYB4 is a negative regulator that improves stress tolerance59, and MYB44 and RVE1 can regulate stress resistance60. WRKY33 and WRKY41 regulate phytohormone levels and responses to biotic and abiotic stress61,62. Therefore, GA improves anther cold tolerance by regulating stress-responsive genes.

Materials and methods

Plants, treatment, and sampling

“Zhipi” almond growing in the Plant Resources Garden of the Xinjiang Academy of Agricultural Sciences (Luntai, Xinjiang, 41° 46′ 58′′ N, 84° 13′ 24′′ E) was selected as the study material. The following samples were collected from early winter to spring between 9:00 and 10:00 A.M.: ZP1 (“Zhipi” at the meiosis stage in the field, January 1), ZP2 (“Zhipi” at the tetrad stage in the field, February 1), ZP3 (“Zhipi” at the 1-nucleus stage in the field, March 1), and ZP4 (“Zhipi” at the mature 2-nucleus stage in the field, March 28). ZP3 flowers were more sensitive to cold and had higher bioactive GA levels than flowers of other stages, so ZP3 flower buds were chosen for further detailed experiments. ZP3 flower buds were treated with exogenous hormones, and 600 branches were cut from 3 trees on March 1, treated at −12 °C until the relative sensitivity of conductivity in the buds reached approximately 50%, and then maintained in a 4 °C climatic cabinet for 24 h. The treated samples were equally divided into three groups: a 100 μmol/L GA3-treated group, a CK group, and a 100 μmol/L PP333-treated group. The branches were fixed in flower putty containing 1/2 MS+5% sucrose hydroponic solutions in a climatic cabinet for 3 days; thereafter, the anthers were collected from 100 branches and stored in liquid nitrogen28. The other branches were cultivated to bloom in a climatic cabinet; their mature anthers were collected for pollen quantification and pollen viability assessment.

Photographs and sections of anthers

Photographs of anthers were taken on a Nikon SMZ-250 stereomicroscope. Paraffin sections of anthers were dyed with toluidine blue, and photographs were taken on a Nikon 80i transmission microscope. Fifty-nanometer-thick anther sections were cut with an LKB-8800 ultrathin slicer, stained with uranyl acetate-lead citrate, and observed on an H-600 transmission electron microscope. Complete pollen samples were dyed with a 5% DAPI solution for 30 min in the dark and observed on a Nikon 80i transmission fluorescence microscope.

Measurement of pollen indexes

Pollen was collected at the time of flowering, and the viability of 1000 pollen grains was tested on a medium containing 10% sucrose and 1% agar powder in a 25 °C incubator for 12 h. The number of pollen grains per anther was counted under a microscope41.

Measurement of different phytohormones

Anthers (1.00 ± 0.05 g, stored in a −80 °C freezer) were accurately weighed and ground in liquid nitrogen; 10 ml of isopropanol-hydrochloric acid extraction buffer was added to the anther powder, and the mixture was shaken at 4 °C for 30 min. Then, 20 ml of dichloromethane was added to the mixtures, which were shaken at 4 °C for 30 min. The treated anthers were next centrifuged at 13,000 r/min for 5 min at 4 °C to obtain the lower organic phase, and the organic phase was dried with nitrogen in the dark, dissolved in 400 μl of methanol containing 0.1% formic acid, and filtered through a 0.22-μm filter to detect the levels of GA compounds, IAA, JA, ABA, tZ, and SA by HPLC-MS41.

RNA isolation, sequencing, and qRT-PCR

Total RNA was isolated from almond anthers with Power SYBR® Green PCR Master Mix (Applied Biosystems® Cat: 4367659) and quantified using BIO-RAD CFX Connect™ (USA). The expression levels determined by qRT-PCR were analyzed based on the average mean of three biological repeats. The quantity of RNA in each sample was estimated with a spectrophotometer and by agarose gel electrophoresis, and samples with higher quality were selected for sequencing on the Illumina PE150 platform; the RNA-Seq data had three biological repetitions. Twenty genes were selected to design primers (Table S3) and validate the RNA-seq data (Table S2), and the AcActin1 gene was used as an internal standard for the normalization of data to calculate relative fold differences based on the comparative cycle threshold (2−ΔΔCT) values4. All the experiments in this study had more than three biological replicates.

Module–trait relationship map, heatmap, and protein interaction map

A module–trait relationship map was produced by weighted correlation network analysis based on the FPKM values of the genes, phytohormone levels and growth rate per 1000 anthers. If one variable moved proportional to another variable, there was a positive correlation (r > 0); in contrast, if variables opposed one another, they displayed a negative correlation (r < 0). The heatmaps of the genes were generated with HemI 1.0 based on the FPKM values of the genes and were refined with Photoshop CS5. The protein-protein interaction maps were produced with the STRING database by referring to the data for Arabidopsis thaliana.