Heat-stress-induced ROS in maize silks cause late pollen tube growth arrest and sterility

Summary The reproductive phase of plants is highly sensitive to ambient temperature stresses. To investigate sensitivity of female reproductive organs in grass crops during the pollination phase, we exposed the elongated stigma (silk) of maize to ambient environment at the silking stage. Moderate heat stress causes cell death of silk hair cells but did not affect early pollen tube growth inside the silk. Late pollen tube growth arrest was observed, leading to sterility. Heat stress causes elevated levels of reactive oxygen species (ROS) in silks, whose levels can be reduced by scavengers partly restoring pollen tube growth and fertility. A number of biological processes including hydrogen peroxide catabolic processes and bHLH transcription factor genes are downregulated by heat stress, while some NAC transcription factor genes are strongly upregulated. In conclusion, this study now provides a basis to select genes for engineering heat-stress-tolerant grass crops during the pollination phase.

(C-D) Fluorescent microscopy of aniline blue stained silks at the 1 to 2 cm proximal region 24 hours after pollination (24 HAP).Silks (3 days after emergence) after 24 hours HS (HS 24h) were pollinated, then kept at NS conditions after pollination (NS AP) (C), or HS exposure for another 4 hours after pollination (D).Red arrowhead indicates a pollen tube in the transmitting tract of silks.Scale bars: 100 μm.
(E-F) Fluorescent microscopy of aniline blue staining of ovule section which contains the female gametophyte and the micropylar region at 24 HAP.Silks (3 days after emergence) after 24 hours HS (HS 24h) were pollinated, then kept at NS conditions after pollination (NS AP) (E) or exposed for another 4h HS after pollination (F).Red arrowhead indicates ae pollen tube at the micropylar region.Scale bars: 50 μm.

Figure S1 .Figure S2 .
Figure S1.Cell death didn't occur within observed time periods under control conditions, related to Figure 1.(A-C) Confocal microscopy of maize silk hairs stained with fluorescein diacetate (FDA) and counter stained with propidium iodide (PI).Maize silks (3 days after silk emergence) at control conditions (NS) (A), after 24 hours (B) and 48 hours (C) without heat stress (HS).Percentages are given for silk hairs lacking dead cells.Scale bars: 50 μm.DIC, FDA and PI channels were merged.(D) Percentage of silk hairs showing cell death (PI-stained nuclei).Letters on columns indicate significantly associated categories.P > 0.05.

(Figure S3 .
Figure S3.Maize silks exposed to heat stress show inhibition of late pollen tube growth, related to Figure 3. (A-B) Fluorescent microscopy of aniline blue stained silks 1 hour after pollination (1 HAP), silks (3 days after emergence) after 24 hours HS treatment (HS 24h) were pollinated with NS pollen, then kept in NS condition after pollination for 1 hour (NS AP) (A) or HS exposure for another 1 hours after pollination (HS 1h AP) (B).Scale bars: 100 μm.

Figure S4 .Figure S5 .Figure S6 .AFigure S7 .BFigure S8 .BFigure S9 .
Figure S4.ROS levels are increased in silks under heat stress and ROS scavengers reduce their levels, related to Figure 4. (A-B) DIC microscopy of nitroblue tetrazolium (NBT) stained silks sprayed with mock solution (A) or sprayed with combined MnCl 2 and SOD solution (B).Silks (3 days after emergence) were exposed to HS for 24 hours (HS 24h).(C) Quantification of mean grey values of NBT stained silks.Data includes 3 biological replicates.Letters indicate significance categories.P < 0.01 by ANOVA test.(D-E) DIC microscopy of 3,3′diaminobenzidine (DAB) stained silks sprayed with mock solution (A) or sprayed with MnCl 2 /SOD solution (B).Silks (3 days after emergence) were exposed to HS for 24 hours (HS 24h).Scale bars: 100 μm.(F) Quantification of mean grey values of DAB stained silks.Data includes 3 biological replicates.Letters indicate significance categories.P < 0.01 by ANOVA test.(K) Quantification of relative fluorescence intensity of silks with a H2DCFDA probe.Letters indicate significance categories.P< 0.01 by one-way ANOVA-TUKEY test.(L-N) DIC microscopy of DAB stained silks sprayed with mock solution (L) or sprayed with ascorbic acid (ASC) solution (M) or MnCl 2 /SOD solution (N) during 48h HS treatment.Scale bars: 100 μm.

Table S2 . Log2FC values of peroxidase (POD) genes, related to Figure 6.
Gene identifier (Gene ID) and BaseMean (average of normalized count values divided by size factors taken over all samples) values are provided.Log2FC indicates transcript's expression level changed between HS and NS groups.P-value indicates test for transcripts between HS and NS groups and padj the adjusted P-value for multiple testing of transcripts.

Table S3 . Top 50 DEGs from enriched TF families, related to Figure 7.
Gene identifier (Gene ID), TF family as well as BaseMean (average of normalized count values divided by size factors taken over all samples) values are provided.Log2FC indicates transcript's expression level changed between HS and NS groups.P-value indicates test for transcripts between HS and NS groups and padj the adjusted Pvalue for multiple testing of transcripts.