High ambient temperature alters the root system architecture with a reduction in the number of emerged lateral roots compensated by their increased elongation
Studying root development in petri dishes has limitations, including a limited growth area, root illumination, and the absence of soil. To overcome these challenges in in vitro plant cultivation, we used a light-isolated rhizotron system that allows plants to grow in soil for non-invasive, image-based root phenotyping. To investigate the effects of temperature on root morphology and the role of the PhyB-PIF4 pathway, we phenotyped Col-0, phyb, 35S::PIF4, and pif4 plants. Lateral and adventitious roots, total root length, primary root length, maximum length of the four longest lateral roots, and root area were measured throughout growth. Root growth patterns responded differently to warm temperatures among genotypes (Fig. 1a). hAT significantly promoted root elongation in Col-0 and pif4, but had little effect on phyb and 35S::PIF4 plants (Additional file 1 - Fig. S1). All plants initially showed increased relative root elongation at hAT (Fig. 1b). However, by the third week of development, root growth was slower at hAT compared to nAT in all genotypes (Fig. 1b). Overall, no significant differences were detected at the final time point (Additional file 1 - Fig. S1), consistent with previous work using a TGRooZ device that mimics natural conditions [46]. hAT had no significant effect on the overall root growth rate of the genotypes studied. In hAT, 35S::PIF4 showed a slightly lower root growth rate of 1.29 cm/day, in contrast to the other genotypes (1.41 to 1.6 cm/day) during the entire measurement period. This difference in growth rates resulted in a smaller average root depth for 35S::PIF4 throughout its growth (Table 1; Additional File 1 - Fig. S2). Moreover, during the initial growth phase (between 10 and 16 days after sowing), at nAT, no significant difference in the relative root growth rate were observed among the genotypes. At hAT, 35S::PIF4 showed a lowest significant root growth rate of 1.42 cm/day. During the second growth phase (between 18 and 21 days after sowing), 35S::PIF4 roots were growing significantly slower than Col-0 at both nAT (1.39 cm/day vs. 1.88 cm/day) and hAT (1.81 cm/day vs. 2.39 cm/day) (Fig. 1b; Additional File 3 – Table S2).
Lateral root formation was inhibited at hAT (Fig. 1c). Wild-type, phyb, and pif4 plants showed reduced lateral root density (number of emerged lateral roots per cm of primary root) at hAT. However, lateral root density was not affected in 35S::PIF4 plants at hAT (Fig. 1c; Additional File 2 - Table S1). At nAT, Col-0 and pif4 plants had 2.2 and 2.11 lateral roots per cm of primary root, respectively. In contrast, phyb and 35S::PIF4 plants produced fewer lateral roots, averaging 1.5 and 1.7 lateral roots per cm of primary root, respectively. At hAT, the number of lateral roots in wild-type plants was similar to that of phyb and 35S::PIF4 at nAT. Although the plants had fewer emerged lateral roots, hAT promoted their elongation in all the genotypes (Fig. 1d). There may be a trade-off between the number of lateral roots and their length. All the genotypes increased the average length of the four longest lateral roots with Col-0 and pif4 seedlings being the most affected and 35S::PIF4 and phyb being the least sensitive to hAT (Fig. 1a,d; Additional File 2 - Table S2). The opposite effects of hAT on the number of lateral roots and their length did not significantly affect the total root length and root area between nAT and hAT (Additional File 1 – Figs. S2 and S3). However, these values were significantly lower for phyb and 35S::PIF4 genotypes under both conditions, resulting in a reduced root system compared to wild-type plants. Furthermore, temperature increase promoted the induction of adventitious roots in all the genotypes studied. 19% of the wild-type and phyb plants produced adventitious roots at hAT, while this value decreased to about 5% for the PIF4-modified genotypes. These changes in (lateral) root length and number alter the root system architecture of plants grown at hAT. These results indicate that phyb and 35S::PIF4 plants under both conditions have a reduced root system compared to wild-type plants. They also suggest that the suppression of PhyB at nAT mimics the effects of hAT on the number of emerged lateral roots.
Suppression of PhyB mimics the effects of high ambient temperatures on Arabidopsis shoot architecture
To understand how hAT affects shoot development and photosynthetic efficiency through the PhyB-PIF4 pathway, we studied eight lines: 35S::PIF4, phyb-9, pif3-7, pif4-2, pif7-1, pif7-2, pif3-3 pif7-1, and pifq (pif1-1 pif3-7 pif4-2 pif5-3) mutants. We quantified the effects of hAT on plant growth by measuring the rosette area from 9 to 39 days after sowing, when the plants reached their final rosette size (Fig. 2, Additional File 1 - Figs. S4, S5). The phyB and 35S::PIF4 plants exhibited delayed rosette expansion, starting at 26 days after sowing, while the other genotypes expanded from 22 days after sowing (Fig. 2a, Additional File 1 - Figs. S5). At nAT, wild-type plants had the largest area (40 cm²), whereas 35S::PIF4 and phyb plants were smaller (20 cm² and 10 cm², respectively) (Additional File 1 - Figs. S4, S5). Other genotypes (pif3, pif7, and pifq) produced plants with intermediate rosette areas. This is a consequence of a significant reduced growth rate between 20 and 27, and between 28 and 33, days after sowing in 35S::PIF4 and phyb plants. It is noteworthy that the phyb plants stopped expanding after 27 days (Fig. 2b; Additional File 1 - Fig. S5). Wild-type plants were significantly sensitive to hAT, with a reduced growth rate and a final area of 15 cm² (Additional File 1 - Fig. S5). In contrast, 35S::PIF4, pif3, pif4, and pifq maintained a stable growth rate between 20 and 27 days (Fig. 2a) but pif3, pif4, and pifq slowed down their growth rate after 28 days (Fig. 2b). The final rosette area was about 20 cm² for pif4, pif7-1, pif7-2, and pif3 pif7 plants. The phyb and 35S::PIF4 plants showed the smallest area with only 5 cm². The wild type, pif3, and pifq showed an intermediate size of 15 cm² (Additional File 1 - Fig. S5).
To analyze the effects of hAT on shoot branching, we measured the number of primary branches emerging from rosette leaves in all genotypes under both growth conditions. While most genotypes produced an average of about five branches under normal conditions, phyb and 35S::PIF4 plants produced an average of three branches. When exposed to hAT, branch production decreased in almost all genotypes. The phyb and 35S::PIF4 plants produced an average of two branches, while the other genotypes produced an average of three branches, mirroring the performance of phyb and 35S:PIF4 under nAT. Notably, the pif3 pif7 and pif7 plants appeared to be resilient to the hAT, roughly maintaining their branch production. At hAT, they outperformed other genotypes, producing an average of 4 branches (Table 2).
The inflorescence growth pattern was affected in hAT (Fig. 3; Additional File 1 - Fig. S4). In nAT, phyb plants flowered at 25 days, earlier than the other lines (29 days). The first flowers of the primary inflorescence stem opened between 25 and 29 days in phyb and between 29 and 36 days in the other genotypes. Consequently, phyb inflorescence stems were longer than Col-0 stems during their growth period, e.g., until 36 days, when both genotypes reached a comparable height. However, the growth rate of the phyb primary inflorescence stem was significantly lower than that of the wild-type stem (Figs. 3c and 3d; Additional File 3 - Table S3). The phyb mutant also stopped flowering earlier, at 44 days, compared to the other genotypes, which continued flowering until 49 days (Fig. 3a). All genotypes grew to a total height of 35 cm by the last observation point at 49 days (Fig. 3a). hAT stimulated early initiation of inflorescence stem elongation in all genotypes at 23 days, similar to that observed in phyb plants grown under nAT (Figs. 3a, 3b). In the primary inflorescence stem, flowers opened around 27-30 days in hAT. Plants reached their maximum growth earlier, at 41 days, as indicated by the significantly reduced growth rate in hAT in wild-type, pif7-1, and pif3 pif7 plants (Figs. 3c, 3d; Additional File 3 - Table S3). This resulted in a shorter final height ranging from 13-38.9 cm (35S::PIF4 stems being the shortest) at hAT, while this value corresponds to 18-43.8 cm at nAT (Figs. 3a, 3b). The main inflorescence stem growth rate was insensitive to temperature changes throughout the entire flowering period in phyb, 35S::PIF4, pif3, pif4, pif7-2, pif3 pif7, and pifq plants (Fig. 3d), and only at the start of the flowering period in pif7-1 plants (Fig. 3c).
Ambient temperature has a moderate impact on plant health but modulates photosynthetic parameters
Since hAT affects different characteristics of plant growth, we wondered how these changes would affect the reflectance profile and pigment content of the plant. Hyperspectral imaging in the visible and near infrared (350-900 nm wavelength, VNIR) measures the light reflectance of plant leaves. It is an important indicator of plant health status [47, 48]. In our study, we measured VNIR parameters, including the Normalized Difference Vegetation Index (NDVI), Optimized Soil-Adjusted Vegetation Index (OSAVI), Photochemical Reflectance Index (PRI), Modified Chlorophyll Absorption Ratio Index 1 (MCARI1), Structure Insensitive Pigment Index (SIPI), and Plant Senescence Reflectance Index (PSRI).
In nAT, NDVI increased with age until 29 days after sowing for all genotypes and remained stable until the end of the measurements at 34 days (Additional File 1 - Fig. S6a; Additional File 3 - Table S6). hAT reduced the NDVI in all genotypes ranging from 0.68 to 0.78, especially at later growth stages (22-28 days after sowing) (Additional File 1 - Fig. S6a). In both nAT and hAT, NDVI had lower values for phyb and 35S::PIF4 with values in nAT (an average of 0.74) being comparable to NVDI values (an average of 0.82) of the other genotypes in hAT. OSAVI, which is designed to mitigate the effects of soil on NDVI, mirrored the trends observed in NDVI (Additional File 1 - Fig. S6b; Additional File 3 - Table S6). These two parameters are indicators of plant vegetative health [49, 50]. Therefore, it can be concluded that both the suppression of PhyB activity and hAT affect the vegetative vitality of the plant.
PRI and PSRI parameters were mostly not significantly affected by the different ambient temperatures for all genotypes (Additional File 1 - Figs. S6c, S6d; Additional File 3 - Table S6). PRI values decreased with the plant age, whereas the opposite was observed for PSRI, which measures plant senescence based on the ratio of carotenoids to chlorophyll. Again, phyb and 35S::PIF4 plants had lower PSRI values than wild type at nAT and hAT. The SIPI parameter is sensitive to chlorophyll and carotenoid content [51] and MCARI1 parameter is associated with the chlorophyll content in plant leaves [52]. Both values increased as the plants aged at nAT and hAT (Additional File 1 - Figs. S6e, S6f; Additional File 3 - Table S6). All other genotypes, except 35S::PIF4 and phyb, had reduced SIPI values at hAT. The 35S::PIF4 and phyb plants had lower SIPI values at nAT and did not respond to hAT. A similar trend was observed for the MCARI1 parameter.
We applied chlorophyll fluorescence imaging to assess the efficiency of the plants to use the light energy for photosynthesis in the studied genotypes at nAT and hAT. The parameter QY-max (FV/FM) indicates the maximum quantum efficiency of the photosystem II (PSII) photochemistry. QY-max of wild-type plants increased steadily with age, with values ranging from 0.79-0.84 for nAT and 0.79-0.82 for hAT (significant difference only between 14 and 32 days after sowing). In nAT, the QY-max values for phyb and 35S::PIF4 plants were lower than in the wild type. Interestingly, phyb recovered to wild-type QY-max values after two weeks of cultivation at nAT (Additional File 1 - Fig. S7a). At hAT, QY-max values increased with age for all genotypes, except 35S::PIF4 and pif3 (Additional File 1 - Fig. S7a). Photosynthetic efficiency was also measured in light-adapted plants. In particular, the parameters QY-Lss (PSII operating efficiency), and qP (photochemical quenching coefficient) [53] displayed significantly higher values at hAT for all the genotypes, corresponding to those of 39-day-old plants grown at nAT for both low and high light saturation point (Lss1 and Lss4) (Additional File 1 - Fig. S7c-f). For the two light intensities at hAT, the age of the plants did not impact the values of the two parameters. Non-photochemical quenching (NPQ) assesses the damage to photosystems caused by various environmental stressors [54]. All the genotypes exhibited lower NPQ values at hAT, indicating the negative impact of the high ambient temperature on the photosystem activity (Additional File 1 - Figs. S7g, S7h). Compared to the wild type, the phyb and 35S::PIF4 plants showed elevated NPQ values at nAT and hAT.
A correlative response to hAT in vegetative organs was observed
To explore potential correlations for the response to hAT among different plant organs, we utilized correlation matrices (Fig. 4). These matrices display correlations with p-values below the significance threshold of 0.05, indicating statistically significant relationships between the relative responses to hAT in the different organs. During vegetative growth (Fig. 4a), a positive correlation (0.96) was observed between the NDVI parameter and the length of the inflorescence stem, highlighting the effectiveness of the NDVI parameter in indicating vegetative growth dynamics. A robust positive correlation (0.94) was also noted between inflorescence stem growth rate and rosette area for their response to hAT, suggesting mutual interdependence between these traits. Notably, a negative correlation (-0.62) was observed between lateral root density and lateral root length, hinting at a potential trade-off mechanism governing root development.
PhyB influences the response of reproductive tissues to hAT
We have used Col-0, phyb, pif4, pifq, and 35S::PIF4 plants to investigate whether the PhyB-PIF4 pathway regulates thermomorphogenesis during reproductive development. To ensure a similar fitness of the plants at the reproductive stage, plants were exposed to hAT after the first flower bud appearance and maintained at hAT until the end of their growth.
Effects of hAT on anthers
Anthers were collected at 7 and 9 days after the development of the first flower (DAFD) on the primary inflorescence stem. In nAT, we did not observe any abnormality in the different lines. In hAT, the wild type, phyb, and 35S::PIF4 lines were affected to different degrees. At 7 DAFD, 4.65 % of the wild-type anthers were aborted, while this percentage reached 23.40 % and 11.43 % for the 35S::PIF4 and phyb lines, respectively. Interestingly, these percentages increased to 7.81 %, 34.82 %, and 29.27 %, respectively, at 9 DAFD when plants were subjected to prolonged hAT. Notably, only the phyb mutant showed a highly significant increase in this trend (Table 3). This observation suggests that the phyb plants may become increasingly sensitive to hAT as they progress through later developmental stages. Additionally, we observed that pif4 and pifq anthers were more resistant to hAT than wild type, with abortion rates of only 1.11 % and 1.44 %, respectively, at 9 DAFD. Our results suggest that suppression of PhyB, resulting in PIF4 activation, worsens the negative effect of hAT on anther development.
Effects of hAT on mature ovules
The same plants were analyzed to determine the effect of hAT on ovules. In nAT, 17.9 % and 16.1 % of phyb and 35S::PIF4 ovules, respectively, were defective, whereas the other lines had between 5.4 % and 9.4 % defective ovules (Fig. 5; Table 4; Additional File 2 – Table S4). Notably, only phyb and 35S::PIF4 lines were defective in the fusion of the central cell nuclei (Fig. 5c; Additional File 2 – Table S4). At hAT, all genotypes exhibited the same types of defects, predominantly a collapsed embryo sac (lacking synergid, egg cell, and central cell structures), collapsed synergids, and unfused central cell nuclei (Fig. 5a-d). Although the types of ovule defects were consistent across genotypes, the percentage of these defects varied (Additional File 2 – Table S4). 35S::PIF4 and phyB ovules were hypersensitive to hAT, producing 84.3 % and 62.6 % defective ovules, respectively (Table 4). In contrast, these percentages were only 30.6 % and 27.6 % in the wild-type and pif4 lines, respectively. Interestingly, more ovules (45.9 %) were defective in pifq than in pif4 (27.6 %), suggesting that other PIFs (such as PIF3, PIF5, or PIF7) may play a synergistic role in this response in ovules. Based on these results, we hypothesize that repressing PhyB expression mimics the temperature effects observed in the wild type during ovule development.
To better understand what would be the molecular mechanism behind the physiological response of Arabidopsis ovules to hAT, we performed a transcriptomic analysis of the gynoecium from 7 DAFD flowers at stage 11-12 (pre-anthesis, ovules at FG7) of Col-0, phyb and 35S::PIF4 plants grown under nAT and hAT. More than 40 million reads were obtained from each sample (Additional File 2 – Table S5), with an average of 45 % GC content. RNA-seq data received a high quality score by the Phred of 98 for Q20 and 94 for Q30 in average.
An overlapping transcriptional response is observed between hAT wild-type pistils and nAT-grown phyb and 35S::PIF4 pistils
The analysis of differentially expressed genes (DEGs) in the different samples and conditions revealed different patterns. In response to hAT, the wild-type pistils had 8,485 DEGs (5,032 up-regulated and 3,453 down-regulated). A lower number of DEGs in phyb and 35S::PIF4 pistils, 1,862 and 2,612 genes, respectively, were distributed as 1037 and 2062 up-regulated genes, and 825 and 550 down-regulated genes, respectively (Additional File 1 - Fig. S8a; Additional File 4 – Table S1). Notably, all genotypes responded to hAT with upregulation of gene expression.
The phenotyping analysis indicated that the phyb and 35S::PIF4 plants at nAT behaved as Col-0 at hAT. Therefore, we compared the DEG patterns of the wild-type pistils in response to hAT with those of phyb and 35S::PIF4 pistils at nAT. The number of up- and downregulated DEGs in these comparisons was very similar (Additional File 1 - Fig. S8b; Additional File 4 – Table S1). Venn diagrams analyze the overlap of the up and down DEGs in the same comparisons. In response to hAT, 10 % (542 genes) of the upregulated genes from wild-type pistils were also upregulated in 35S::PIF4 and phyb pistils at hAT, whereas only 3 % (121 genes) of the downregulated genes from wild-type pistils were also downregulated at hAT in the two mutants (Additional File 1 - Figs. S8c, S8d; Additional File 4 – Table S1). However, only 3.7% of the genes upregulated in the wild-type pistils in response to hAT were also upregulated in both phyb and 35S::PIF4 pistils at nAT. The majority of the upregulated DEGs (62 %) in wild-type pistils at hAT were also found to be upregulated in 35S::PIF4 pistils at nAT (Additional File 1 - Fig. S8e; Additional File 4 – Table S1). In addition , almost half of the genes downregulated in the wild-type pistils at hAT (46 %) were downregulated genes in phyb and 35S::PIF4 pistils at nAT (Additional File 1 - Fig. S8f; Additional File 4 – Table S1). Wild-type Arabidopsis pistils (and ovules) developed at hAT showed pronounced transcriptional changes, mostly as upregulation, with a substantial overlapping regulation with phyb and 35S::PIF4 pistils developed at nAT. This suggests that the Arabidopsis response to hAT during pistil development may involve signaling pathways dependent on the PhyB and PIF regulators.
Gene ontology analysis identified biological processes affected by hAT and PhyB-PIF4 signalling in pistils
Gene Ontology (GO) functional annotation analysis using was performed for up- and downregulated DEGs in wild-type pistils from plants grown on hAT, and 35S::PIF4 and phyb pistils from plants grown on nAT to determine whether the hAT response in wild-type pistils shares GO patterns with the response in pistils from plants defective in the PhyB pathway (Fig. 6; Additional File 4 – Table S2).
Cell division rate is known to be dependent on ambient temperature [55]. Several GO terms related to cell division, cell cycle, DNA replication, and mRNA processing were enriched among the commonly upregulated DEGs. These processes are known to be critical during pistil and ovule development. Indeed, GO terms associated with megagametogenesis, ovule, embryo sac, and flower development and the transition to the reproductive phase in the meristem were among the commonly upregulated DEGs (Fig. 6a). Among the GO terms related to fertilization and reproduction, recognition of pollen, (regulation of) pollen growth and pollen development were enriched. Genes involved in pollen tube growth were specifically upregulated by hAT in the wild-type pistils, whereas genes involved in pollen germination were enriched only in 35S::PIF4 pistils (Fig. 6a). This suggests that both hAT and the PhyB-PIF4 pathway may influence the expression of genes involved in ovule development as observed in Fig. 5, and that fertilization processes dependent on pollen tube growth and guidance may be specifically affected by hAT in wild-type pistils.
Surprisingly, GO terms related to the responses to hormones and abiotic stresses were found to be downregulated (Fig. 6b). Responses to auxin and ethylene were downregulated in all sample comparisons. However, GO terms associated with brassinosteroid, gibberellin, abscisic acid, and jasmonic acid were exclusively downregulated in 35S::PIF4 pistils, which may explain the more pronounced phenotypic response of 35S::PIF4 pistils to hAT during ovule development (Table 4; Additional File 2 - Table S4) . Furthermore, GO terms related to cold and light stress responses, photosynthesis, protein translation, and metabolism were generally enriched among the downregulated genes in all three samples (Fig. 6b).
The expression profile of the phyb and 35S::PIF4 pistils at nAT for auxin signaling and miRNA processing genes is comparable to that of wild-type pistils at hAT
Hierarchical clustering analysis of the expressed genes identified two major clusters among the top 100 DEGs in Col-0 hAT pistils compared to Col-0 nAT pistils (Additional File 1 - Figure S10; Additional File 4 – Table S3), the DEGs involved in the auxin signaling pathway (Fig. 7a; Additional File 4 – Table S3) and in miRNA biogenesis (Fig, 7b; Additional File 4 – Table S3). One cluster consists exclusively of the wild-type pistils from plants grown in nAT. The second cluster includes the pistils from phyb and 35S::PIF4 plants grown in nAT and hAT, as well as from wild-type plants grown in hAT. Similar to what was observed during our phenotyping analysis, these results indicate that the response to hAT and to the PhyB-PIF4 pathway share a gene regulatory network.
PIF4 binds to the promoters of several miR156 genes to repress their expression, resulting in the accumulation of the miR156 target transcripts, the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes [56]. SPL will then regulate plant growth in response to shade and warm temperature. The module miR156/SPL9 regulates the thermomorphogenetic response of the hypocotyl by mitigating its sensitivity of auxin [57]. Several SMALL AUXIN UP RNA (SAUR) and Aux/IAA genes, as well as AUXIN RESPONSE FACTOR ARF10 and ARF19 are upregulated in the second cluster (Fig. 7a). We also identified MIR156, MIR160, and the miRNA processing AGO1, DCL1 genes to be upregulated in the same cluster, while the MIR156 targets SPL5 and SPL9 were slightly down-regulated (Fig. 7b).
Pollen tube attractants are upregulated at hAT
We also performed a hierarchical clustering for genes related to pollen tube guidance, an enriched GO term category (Fig. 6a; Fig. 7c; Additional File 4 – Table S3). Again, two distinct clusters related to the hAT response were identified. Genes encoding the defensin-like pollen tube attractants CYSTEINE-RICH PEPTIDE (CRP) AtLURE1s and XIUQIU, EMBRYO SURROUNDING FACTORS 1.3 (ESF1.3), EGG CELL SPECIFCs (ECSs), and MYB98, a transcription factor controlling their expression [58, 59], were upregulated in the cluster comprising all pistil samples from plants grown in hAT (Fig. 7c), regardless of genotype.
Changes in YUCCA and TAA1 expression levels in hAT in mature ovules suggest a role for auxin biosynthesis in the response to high ambient temperature
In seedlings, hAT-activated PIF4 enhances the expression of the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1), YUCCA 8 (YUC8) and SAUR genes in the leaves and hypocotyls [8, 20]. TAA1, YUC4 and YUC8 are also expressed in mature ovules at the micropyle cells surrounding the embryo sac [60]. To evaluate the effects of hAT on auxin homeostasis in mature ovules, we analyzed the expression pattern of the three auxin biosynthetic genes. TAA1 is expressed in the micropylar cells in nAT and its expression is altered in hAT (Fig. 8a-c). The TAA1 fluorescence signal was not detected in 49 % of the ovules and was weak in the remaining samples in hAT (Figs. 8b, 8c). YUC4 was strongly expressed in the integuments of mature nAT ovules (Fig. 8d). Different levels of the fluorescence signal intensity were observed for YUC4 in hAT ovules: same expression pattern with reduced signal intensity (19.4 %; Fig 8e), restricted expression domain at the chalazal integuments with weak signal intensity (66.6 %; Fig. 8f), and no signal (13.8 %; Fig. 8g). YUC8 showed no (95.4 %; Fig. 8h) to weak expression in the micropylar cells (4.6 %) in nAT ovules. However, in hAT, YUC8 was highly expressed in the micropylar cells (Fig. 8i). YUC8 is known to be upregulated in hAT in other tissues [8], which is consistent with our observations in ovules. The contrasting expression behavior of YUC4 and YUC8 at hAT suggests an intricate and complex regulatory mechanism in the response to hAT in the ovules.
Effects of hAT on early embryo development
Given the effects of hAT on ovules and the transcriptional changes associated with pollen guidance and its impact on fertilization, we investigated the effects of hAT on seed and embryo development in the same genotypes. Seeds bearing embryos from early developmental stages (one-cell to late globular) were analyzed for embryo patterning defects. In nAT, no significant differences were observed between the different genotypes (Table 5). In hAT, however, all genotypes were significantly affected. No statistically significant differences in the percentage of defective embryos were observed between wild type (40.77 %), pif4 (44.23 %), pifq (41.56 %), and phyb (30.85 %). Only 35S::PIF4 appeared to be resistant to growth at hAT with a significantly lower embryonic defect rate of 21.95 % (Table 5). A variety of embryonic defects have been observed, including an excess of cell divisions within the proper embryo or suspensor, irregularities in the size of the hypophysis cell, and a reduction in the length of the suspensor (Figs. 5f-h; Additional File 2 - Table S6). A shorter suspensor was observed in all the genotypes for hAT (Fig. 5h). In nAT, the suspensor of the 35S::PIF4 embryos was longer (111 μm) than the wild-type suspensor (97.18 μm). However, this difference disappeared in hAT, suggesting that the 35S::PIF4 embryos were the most affected by temperature variation for suspensor growth (Fig. 5h; Additional File 2 – Table S6). These results suggest that ectopic overexpression of PIF4 may confer a minor temperature resistance during embryogenesis.
hAT-induced changes in seed traits
Dry seeds harvested from the same plants flowering at nAT and hAT were phenotyped using the Boxeed robot. We focused on four seed traits: number of seeds produced per silique, seed shape, seed size, and seed weight (Fig. 9; Additional File 3 – Tables S8 and S9). Elevated ambient temperatures led to an increase in seed area in all the genotypes, with the production of larger viable seeds and smaller misshapen seeds (Fig. 9a). Seed area increased by 34.74 % in Col-0, 31.73 % in 35S::PIF4, 47.83 % in phyb, 25.20 % in pif4, and 47.67 % in pifq (Fig. 9b; Additional File 2 - Table S7). Additionally, seeds produced under warmer conditions were rounder across various genotypes, as assessed by the ratio of the seed length to the seed area. The phyb seeds were the most affected by shape changes in hAT (Fig. 9c; Additional File 2 – Table S8). Evaluation of the number of seeds per silique showed that all genotypes produced fewer but heavier seeds per silique at hAT in all the genotypes (Figs. 9d, 9e; Additional File 2 – Tables S9 and S10). Interestingly, at nAT, phyb seeds were by 25% heavier than wild-type seeds (Fig. 9e; Additional File 2 – Table S10). The higher seed weight observed in seeds developed at hAT suggests a possible adaptive strategy in which plants may favor the production of nutrient-rich seeds rather than a greater number of seeds. However, phyb plants grown on nAT and wild-type plants grown on hAT produced a comparable number of seeds, precisely 42.14 and 47.75 seeds per silique for a comparable weight, 2.33 mg and 2.26 mg per 100 seeds, respectively.
The correlation of the hAT response in reproductive tissues
A correlative analysis of the effects of hAT on reproduction showed that seed number and the increased number of embryo defects and pollen defects were significantly negatively correlated (-0.92 and -0.63, respectively). Seed number and seed weight were also significantly negatively correlated (-0.71). Surprisingly, pollen defects were positively correlated (0.87) with increased seed weight.