Heat stress at the bicellular stage inhibits sperm cell development and transport into pollen tubes

Abstract

For successful double fertilization in flowering plants (angiosperms), pollen tubes deliver 2 nonmotile sperm cells toward female gametes (egg and central cell, respectively). Heatwaves, especially during the reproduction period, threaten male gametophyte (pollen) development, resulting in severe yield losses. Using maize (Zea mays) as a crop and grass model system, we found strong seed set reduction when moderate heat stress was applied for 2 d during the uni- and bicellular stages of pollen development. We show that heat stress accelerates pollen development and impairs pollen germination capabilities when applied at the unicellular stage. Heat stress at the bicellular stage impairs sperm cell development and transport into pollen tubes. To understand the course of the latter defects, we used marker lines and analyzed the transcriptomes of isolated sperm cells. Heat stress affected the expression of genes associated with transcription, RNA processing and translation, DNA replication, and the cell cycle. This included the genes encoding centromeric histone 3 (CENH3) and α-tubulin. Most genes that were misregulated encode proteins involved in the transition from metaphase to anaphase during pollen mitosis II. Heat stress also activated spindle assembly check point and meta- to anaphase transition genes in sperm cells. In summary, misregulation of the identified genes during heat stress at the bicellular stage results in sperm cell development and transport defects ultimately leading to sterility.

Figure 1.

Heat stress during the unicellular and bicellular stages of pollen development reduces seed set in maize. A) Experimental setup. Maize plants were grown in control conditions (25 °C/21 °C light/dark period) until they reached either the unicellular or bicellular stage of pollen development. Stages were identified according to Begcy and Dresselhaus (2017). At either stage V13 (unicellular; red dotted line) or V15 (bicellular; red line), plants were exposed to moderate heat stress (35 °C/25 °C light/dark period) for 48 h and afterward transferred back to control conditions until maturity. Blue line indicates (NS) conditions. NS cobs were pollinated with either NS pollen (NS × NS), (B) HS pollen at the unicellular stage (♂HS Uni × ♀NS), or (C) HS pollen at the bicellular stage (♂HS Bi × ♀NS). Images were digitally extracted for comparison. D) Seed set was strongly reduced after pollinating with HS pollen, regardless of the pollen developmental stage in which heat stress was applied. n = 15 for panels B) and C). Data are presented as the mean ± Sd. Two asterisks indicate significant difference at P < 0.001; 1-tailed t test comparing HS (red) with NS (blue) samples.

Figure 2.

Heat stress at the unicellular and bicellular stages accelerates pollen development. A) Pollen stained with FDA after NS and (B) HS treatment at the unicellular stage. C) Quantification of enzymatic activity of pollen shown in A) and B). D) Pollen stained with FDA after NS and (E) HS treatment at the bicellular stage. F) Quantification of enzymatic activity of pollen shown in D) and E). G) DAPI staining of NS and (H) HS pollen at the unicellular stage. I) Quantification of pollen shown in G) and H). Steel blue, undefined stage; artic blue, unicellular stage; azure blue, bicellular stage; and teal blue, tricellular stage of maize pollen development. J) DAPI staining of NS and (K) HS pollen at the bicellular stage. L) Quantification of pollen shown in J) and K). For description, see I). Data are presented as the mean ± Sd. n = 400 to 500. Scale bars = 50 μm. Two asterisks indicate significant difference at P < 0.001; 1-tailed t test comparing HS (red) with NS (blue) samples.

Figure 3.

Heat stress applied at the unicellular but not at the bicellular stage impairs pollen germination capabilities. A) In vitro germination assays of pollen isolated from NS and (B) HS plants at the unicellular stage. C) In vitro germination rate of pollen shown in A) and B). D) Germination speed of NS and HS pollen at the unicellular stage. E) Aniline blue staining of NS and (F) HS pollen at the unicellular stage germinating on papilla hair cells. G) In vivo penetration rate of pollen shown in E) and F). H) In vitro germination assays of pollen from NS and (I) HS plants at the bicellular stage. J) In vitro germination rate of pollen harvested from NS and HS plants at the bicellular stage shows no germination rate differences. K) Germination speed and (L) pollen tube length of NS and HS pollen at the bicellular stage. M) Aniline blue staining of NS and (N) HS pollen at the bicellular stage germinating on papilla hair cells shows normal pollen tube penetration. O) In vivo penetration rate of pollen shown in M) and N). Statistically significant differences in germination rate, pollen tube length, and germination speed could not be detected at the bicellular stage between HS and NS conditions. Scale bars = 100 μm. Data are presented as the mean ± Sd. n = 400 to 500. One asterisk indicates significant difference at P < 0.01; 2 asterisks indicate significant difference at P < 0.00; ns indicates no statistical differences. One-tailed t test comparing HS with NS samples.

Figure 4.

Heat stress at the bicellular stage impairs sperm cell development and transport into the pollen tube. Maize sperm cell maker lines containing α-tubulin fused with YFP were grown in control conditions (25 °C/21 °C light/dark period) until they reached the bicellular stage of pollen development. Then, plants were exposed to heat stress (35 °C/25 °C light/dark period) for 48 h. A parallel set of plants was maintained under control conditions. A) Confocal and (B) merged images of NS pollen at the bicellular stage. C) Confocal and (D) merged images of HS pollen at the bicellular stage. E) Quantification of YFP signal intensity of pollen as indicated. F) Confocal images of NS pollen grains showing sperm cells traveling into the pollen tube. G) Sperm cells were kept inside the pollen grain after heat stress was applied at the bicellular stage of pollen development. H) Ratio of sperm cells remaining in pollen grain to those traveling inside the pollen tube. I) Reduction in the number of detectable cytoskeletal-related proteins in NS and HS pollen at the bicellular stage. See Supplementary Table S1 for details. Asterisks indicate a significant difference at P < 0.001; 1-tailed t test comparing NS and HS samples. Data are presented as the mean ± Sd. Scale bars = 50 μm. n = 400 to 500.

Figure 5.

Heat stress during the bicellular stage decreases content of centromeric histones in maize. Marker lines expressing centromeric histones (CENH3) specifically in sperm cells (GEX3p:mRuby3-CENH3) were grown in control conditions (25 °C/21 °C light/dark period) until they reached the bicellular stage of pollen development and then submitted to heat stress (35 °C/25 °C light/dark period) for 48 h. A parallel set of marker line plants was maintained under optimal conditions and used as controls. A) Confocal images showing CENH3 in mature pollen grains from NS and (B) HS plants. Insets show enlarged sperm cells containing CENH3 signals. Insets were magnified 3×. C) Intensity quantification of mRuby3-CENH3 in both conditions. Asterisks indicate a significant difference at P < 0.001; 1-tailed t test comparing NS and HS samples. Data are presented as the mean ± Sd. Scale bars = 50 μm.

Figure 6.

Heat stress at the bicellular stage misregulates replication-associated genes in sperm cells. A) Confocal images of NS and (B) HS sperm cells at the bicellular stage of pollen development. Insets show enlargement of single sperm cells (arrowheads). Insets were magnified 4×. Scale bar = 50 μm. Approximately 5,000 individual sperm cells in each of the 3 biological replicates per condition were used. C) Differential gene expression (base 2 logarithm fold change) in sperm cells harvested at maturity after heat stress was applied at the bicellular stage of pollen development. HS samples relative to control (NS) samples were plotted versus average gene expression levels (i.e. logarithm of mean counts normalized for difference in library sizes). Red color indicates upregulation. Blue color indicates downregulation. Black color indicates no significant transcriptional change. D) TPM values and RT-qPCR analysis show that differential expression of HSP genes in sperm cells was still increased in mature pollen. Asterisks indicate significant difference at P < 0.01; 1-tailed t test comparing HS with NS samples. n = 3 biological replicates, each with 3 technical replicates. Data are presented as the mean ± Sd. E) Gene network analysis of interactions of differentially expressed genes in sperm cells in response to heat stress at the bicellular stage. A threshold of 0.7 edge confidence was used. A detailed list of the genes included in the gene interaction analysis can be found in Supplementary Table S4.

Figure 7.

KP1-CUL1-F-box protein (SCF) E3 ubiquitin ligase complex and spindle assembly check point (SAC) genes are upregulated in sperm cells after heat stress was applied during the bicellular stage of pollen development. A) Induction of the SCF member genes. B) Misregulation of the APC/C complex genes. C) Upregulation of cyclins. D) Illustration of the cell cycle during PM II and its regulation by cyclins and the SCF complex. E) Upregulation of SAC gene members after heat stress. NDC80, component of the kinetochore complex. F) Downregulation of microtubule-associated genes. G) Misregulation of tubulin-associated genes. H) Misregulation of genes associated with mitosis progression during the metaphase-to-anaphase transition. WAPL, wings apart-like protein homolog; RCC1, regulator of chromosome condensation 1; Sister, sister chromatid cohesion 1. One asterisk indicates significant difference at P < 0.01; 2 asterisks indicate significant difference at P < 0.001; 1-tailed t test comparing NS and HS samples. n = 3 biological replicates, each with 3 technical replicates. Data are presented as the mean ± Sd.