(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



Multiple light signaling pathways control solar tracking in sunflowers [1]

['Christopher J. Brooks', 'Department Of Plant Biology', 'University Of California', 'Davis', 'California', 'United States Of America', 'Hagop S. Atamian', 'Schmid College Of Science', 'Technology', 'Chapman University']

Date: 2023-11 

Sunflowers are famous for their ability to track the sun throughout the day and then reorient at night to face east the following morning. This occurs by differential growth patterns, with the east sides of stems growing more during the day and the west sides of stems growing more at night. This process, termed heliotropism, is generally believed to be a specialized form of phototropism; however, the underlying mechanism is unknown. To better understand heliotropism, we compared gene expression patterns in plants undergoing phototropism in a controlled environment and in plants initiating and maintaining heliotropic growth in the field. We found the expected transcriptome signatures of phototropin-mediated phototropism in sunflower stems bending towards monochromatic blue light. Surprisingly, the expression patterns of these phototropism-regulated genes are quite different in heliotropic plants. Most genes rapidly induced during phototropism display only minor differences in expression across solar tracking stems. However, some genes that are both rapidly induced during phototropism and are implicated in growth responses to foliar shade are rapidly induced on the west sides of stems at the onset of heliotropism, suggesting a possible role for red light photoreceptors in solar tracking. To test the involvement of different photoreceptor signaling pathways in heliotropism, we modulated the light environment of plants initiating solar tracking. We found that depletion of either red and far-red light or blue light did not hinder the initiation or maintenance of heliotropism in the field. Together, our results suggest that the transcriptional regulation of heliotropism is distinct from phototropin-mediated phototropism and likely involves inputs from multiple light signaling pathways.

In the current study, we use RNA sequencing to define and compare the transcriptional profiles of sunflowers undergoing heliotropism, phototropism, and autostraightening to better understand relationships between these processes. We find that genes differentially expressed across stems bending towards directional blue light are enriched for auxin-related functions, consistent with previous findings in other species and organs [ 13 ]. However, we find limited overlap between genes and pathways differentially expressed across phototropic and autostraightening stems, consistent with distinct mechanisms regulating these responses. To our surprise, our analysis of genes differentially expressed across solar tracking stems also revealed limited similarity between the transcriptomic signatures of blue light–mediated phototropism and heliotropism. This prompted us to examine gene expression across the stems of plants just initiating solar tracking. Interestingly, genes differentially expressed during the onset of heliotropism are distinct both from those differentially expressed in established solar trackers and in plants bending towards blue light in a growth chamber. Instead, we find that genes implicated in shade avoidance responses have rapidly increased expression on the west sides of stems during the onset of heliotropism, suggesting a possible role for phytochrome photoreceptors in this process. However, depletion of red and far-red or of blue light had little effect on the initiation or maintenance of solar tracking in the field. Collectively, our data suggest that heliotropism is transcriptionally regulated by mechanisms distinct from classic phototropin-mediated phototropism and is likely controlled by multiple light signaling pathways.

One growth process that may incorporate responses to light, autostraightening, and circadian regulation is sunflower heliotropism. Sunflowers have an amazing ability to track the sun throughout the day and reorient to face east in anticipation of sunrise the following morning [ 22 ]. This solar tracking is accomplished by antiphasic growth patterns on the east and west sides of the stem, with the east side growing more during the day and the west side growing more at night. Sunflowers moved from the field into constant environmental conditions with a fixed overhead light will continue oscillatory east–west stem bending movements for several days, suggesting a role for the circadian clock in solar tracking [ 22 ]. Moreover, these tracking movements can be recapitulated in a growth chamber using a simulated moving blue light source, suggesting a role for phototropin-mediated phototropism. Limited gene expression analysis revealed that several homologs of genes involved in auxin signaling are differentially expressed across the east and west sides of heliotropic stems, further suggesting a role for the well-characterized phototropin pathway in this process [ 22 ]. Although the above results suggest heliotropism may be a specialized form of phototropism, this has not previously been rigorously examined.

The circadian system has been shown to modulate plant responsiveness to a wide range of environmental cues, including tropic responses to light [ 22 – 24 ]. A functional circadian clock promotes plant fitness by coordinating internal processes with changes in the external environment [ 25 ]. The plant circadian oscillator is made up of a complex network of transcriptional regulators that control each other’s expression and expression of thousands of output genes [ 26 ]. The circadian clock can generate daily growth patterns, with many plants displaying 24-hour rhythms in stem elongation and leaf movement even when maintained in constant environmental conditions [ 27 ].

While responding to the environment is critical, plants also respond to internal cues to modulate growth. Plants exhibit autotropic control of posture so as to maintain an erect growth habit [ 15 ]. In this process, often called proprioception or autostraightening, portions of a stem bent during a tropic response subsequently straighten in a gravity-independent process [ 16 – 18 ]. The mechanisms underlying this proprioceptive response are still poorly understood; however, it is believed that there is a mechanical sensing component involving the actin–myosin XI cytoskeleton [ 19 ]. It has been proposed that specialized fiber cells with large bundles of actin filaments may act as propriocytes, sensing and/or responding to the stem bending [ 15 , 19 ]. While molecular details remain to be determined, there is evidence that, unlike phototropism, this process does not depend upon an auxin gradient across the straightening stem [ 20 , 21 ].

One of the best characterized growth responses in plants is phototropism. During this process, plants grow or bend either towards or away from a directional light source [ 9 ]. Research has for decades focused on the mechanisms regulating the phototropic bending of embryonic organs such as hypocotyls towards directional blue light [ 7 ]. Work primarily conducted on the Arabidopsis thaliana hypocotyl has revealed that the main photoreceptors mediating this response are 2 related membrane-associated protein kinases, PHOTOTROPIN 1 (PHOT1) and PHOTOTROPIN 2 (PHOT2). When exposed to blue light, interactions between phot1 and phot2 and another membrane-associated protein NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) are disrupted [ 10 ]. Although the signaling pathway is not fully understood, the phototropins and NPH3 are essential for the redistribution of auxin, which is carried out by the polar subcellular redistribution of PIN-FORMED (PIN) auxin transport proteins [ 11 , 12 ]. Auxin redistribution creates an auxin gradient within the hypocotyl, leading to higher auxin levels on the shaded sides of plants compared to the lit sides. In Brassica oleracea hypocotyls, phototropism-induced auxin gradients lead to an induction of multiple genes on the shaded sides of the stems. This up-regulation occurred prior to the growth response, suggesting a role for these auxin-induced genes in phototropic growth [ 13 ]. These genes include members of the SMALL AUXIN UP-REGULATED (SAUR) gene family. A subfamily of these auxin-induced SAUR proteins has been reported to inhibit D-CLADE TYPE 2C PROTEIN PHOSPHATASEs (PP2C-D), which, in turn, promote the phosphorylation and activation of plasma membrane proton ATPases. The subsequent extrusion of protons from cells leads to the acidification of the apoplast and cell expansion [ 14 ].

Plants display an extraordinary ability to alter their growth in response to environmental cues, a phenomenon that has fascinated scientists for hundreds of years. With light being a necessary resource, plants use alterations in light quality, intensity, and direction as cues to direct different growth behaviors [ 1 , 2 ]. This is accomplished using a wide range of photoreceptors, which sense these changes in light and then signal appropriate growth responses. For example, phytochromes are red/far-red photoreceptors that allow plants to sense neighbors and alter growth in an attempt to outgrow competitors in a process called shade avoidance [ 3 ]. Plants can sense UV light with UV RESISTANCE LOCUS8 (UVR8) and blue light with cryptochromes and phototropins [ 4 – 6 ]. These photoreceptors also affect plant form, with UVR8 and cryptochromes playing important roles in photomorphogenesis and the phototropins mediating directional growth towards blue light [ 7 ]. Finally, ZEITLUPE is a blue light photoreceptor that conveys information about the light environment to the plant circadian clock [ 8 ].

Results

Transcriptome analysis implicates auxin signaling in heliotropic growth at night but not during the day We next wished to identify genes with different expression patterns on the east and west sides of heliotropic stems. For most genes, the diel expression patterns across the stem are quite similar, consistent with the near-identical patterns of expression of clock genes on the east and west sides of stems (Figs 2E, 2F and S5). However, when comparing the times of peak expression of transcripts significantly rhythmic on the 2 sides, we found that 1,369 genes have acrophase differences of greater than 3 hours on opposite sides of the stem (Fig 2H and S6 Data). Many of these, for example, XTH23-like, appear to have different waveforms on the 2 sides as well (Fig 2K). We next compared the rhythmic amplitude of transcripts on the east and west sides of stems (Fig 2I and S7 Data). As was true for our phase analysis, most genes were very similar, with only 811 having a greater than 2-fold difference in amplitude across the stem. Some of these transcripts, such as EXPL1-like, have much higher overall expression levels on one side of the stem compared to the other (Fig 2L). We next compared expression across the stem at individual time points to identify genes and pathways associated with heliotropic movement. This analysis revealed relatively few differentially expressed genes early in the day, at ZT0 and ZT4, but a larger number of transcripts differentially expressed in the afternoon and early evening, ZT8 and ZT12. Surprisingly, we found the largest numbers of differentially expressed genes at night, ZT16 and ZT20 (Fig 2J and S8 Data), times when there cannot be unequal illumination across the stem. These data demonstrate there is significant differential expression across the stems of solar tracking plants, but not phased to the times of day we had expected. We previously showed that the east sides of solar tracking stems grow faster than the west sides during the day while the west sides grow faster than the east sides at night [22]. To identify biological processes correlated with these growth patterns, we next performed GO analysis on genes differentially expressed at each time point. Since previous studies, along with our own findings, have suggested involvement of light responses, hormone signaling, and growth-related processes in phototropism [9], we focused on these terms. We found enrichment for blue light and UV responses on the east sides of stems in the early morning (ZT0 and ZT4), as well on the west sides in the early afternoon (ZT8), consistent with the position of the sun relative to plant stems at these times (Fig 2A and 2M). We next considered enriched growth-related GO categories. As expected, we found enrichment for cell wall–related processes at ZT8 and ZT12 on the east side, and at ZT16 and ZT20 on the west side, coinciding with the times of antiphasic growth on the opposite sides of the stem (Fig 2B). Surprisingly, however, auxin signaling and response terms were not enriched among genes more highly expressed on the east side during the day. However, these terms were enriched among genes more highly expressed on the west side in the evening and early night (ZT12 and ZT16) (Fig 2M and S9 Data). These results suggest a potential role for the auxin signaling pathway during nighttime west-to-east reorientation, but not during daytime east-to-west growth.

Gene expression analysis suggests the onset of heliotropism and blue light–dependent phototropism are regulated by distinct pathways We next wondered if the limited similarity between genes differentially expressed during daytime heliotropism and in phototropism was because the latter process is an acute response to light while the former involves the circadian system [22]. While sunflowers grown in a chamber with overhead light do not bend [51], heliotropic-type movements can quickly be induced using directional lights sequentially turned on and off to mimic the movement of the sun [22]. In addition, rhythmic bending can be observed for several days after plants are moved from the field into constant environmental conditions. Thus, while directional light is necessary to initiate tracking, and later to reinforce it, it is not absolutely required for heliotropic growth patterns. We therefore asked whether the onset and continuation of solar tracking are characterized by different transcriptional programs and in particular whether the initial onset is transcriptionally similar to phototropism in a growth chamber. We therefore characterized the growth and gene expression patterns of plants at the onset of heliotropism. To this end, 2-week-old chamber-grown plants were transplanted into the field after dusk and imaged for 3 days. During the first day in the field, sunflowers bent east a small amount in the morning and bent west to a greater degree in the afternoon. To our surprise, these plants reoriented during the first night so that they were inclined to the east before dawn (Fig 4A). These data suggest that heliotropism begins the first day young plants are transferred to the field. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Initiation of heliotropism shows a distinct transcriptional profile and occurs in different light conditions. (A) Stem angles of 2-week old chamber-grown sunflowers transplanted into the field after sunset (mean +/− SEM, n = 17). (B-H) Normalized expression of selected phototropically regulated genes during the first 3 days in the field (mean +/− SEM, n = 3, except ZT64 west, n = 2). Statistical significance calculated using Welch’s t test, * represents qvalue < 0.05; † represents qvalue = 0.063. (B) IAA4-like (Ha412HOChr10g0435441). (C) SAUR12-like (Ha412HOChr07g0303111). (D) IAA4-like (Ha412HOChr01g0004341). (E) PIN7-like (Ha412HOChr01g0035481). (F) PHYB-like (Ha412HOChr02g0079361). (G) HB2-like (Ha412HOChr13g0623611). (H) BIM1-like (Ha412HOChr02g0064141). (I) Wavelengths of light measured inside 3 filter boxes. (J) Zscore-normalized stem angles of sunflowers inside filter boxes after their transfer from growth chamber to field (mean +/− SEM, n = 5). Data from one representative experiment. (K) Percentage of plants with significant rhythmic solar tracking in each of the 3 filter boxes (pvalue < 0.001, n = 22). (L) Percent increase in length of sunflower stems in each filter box (J) from dawn on day 1 to dawn on day 5. The underlying raw data may be found in S13 Data for Fig 4J; in S14 Data for Fig 4A-H; and in S15 Data for Fig 4I, 4K, and 4L. BIM1, BES1-INTERACTING MYC-LIKE1; HB2, HOMEOBOX PROTEIN 2; IAA4, INDOLE-3-ACETIC ACID INDUCIBLE4; PHYB, PHYTOCHROME B; PIN7, PIN-FORMED7; SAUR12, SMALL AUXIN UP-REGULATED RNA12; SEM, standard error of the mean; ZT, Zeitgeber Time. https://doi.org/10.1371/journal.pbio.3002344.g004 Next, we examined transcriptional responses across the stem during these first few days. Two-week-old chamber-grown sunflowers were transplanted into the field as described above, and peels from the east and west sides of the stem were collected every 2 hours during the first day and every 4 hours for 2 more days. RNA was extracted from the stem peels, and gene expression was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). First, we examined expression of genes that were rapidly up-regulated and then quickly down-regulated on the shaded sides of stems during phototropism (Figs 3H and S8). These included homologs of the known Arabidopsis auxin-related genes INDOLE-3-ACETIC ACID INDUCIBLE4 (IAA4), SMALL AUXIN UP-REGULATED RNA12 (SAUR12), and PIN-FORMED7 (PIN7) [52]. Surprisingly, only one of these auxin-related transcripts (IAA4-like (Ha412HOChr10g0435441)) was differentially expressed across the stem at any time point during the first day in the field despite the obvious heliotropic bending during that time (Figs 4B–4E and S8A–S8D). However, at no time did we observe the rapid and one-sided up-regulation of expression these transcripts demonstrate during phototropism in the growth chamber (S8A–S8D Fig). During the second and third days, however, their expression patterns began to mirror those observed in the RNA-seq data generated from plants grown in the field for over a week (S8A–S8D Fig). We next examined the expression of 2 transcripts that undergo rapid and sustained up-regulation on the shaded sides of stems during phototropism (S8E and S8F Fig). These encode homologs of Arabidopsis PHYTOCHROME B (PHYB) and HOMEOBOX PROTEIN 2 (HB2), genes known to be involved in shade avoidance [53,54]. These transcripts are rapidly up-regulated on the west sides of stems 2 hours after dawn; however, this is only true on the first day after transfer to the field (Fig 4F and 4G). A homolog of BES1-INTERACTING MYC-LIKE1 (BIM1), another gene involved in shade avoidance [55], also had increased expression on the west side at ZT2, albeit not at levels reaching statistical significance (Fig 4H). We next examined the expression patterns of these 3 genes in our RNA-seq data of plants undergoing established solar tracking. Only the homolog of HB2 is differentially expressed in this condition, being more highly expressed on the east sides of stems at ZT8 (S8 Data). In contrast to what we observed in plants initiating heliotropism, none of these 3 genes are similarly differentially expressed across the stems of established heliotropic sunflowers early in the day. Together, these data demonstrate that even though plants responding to unidirectional blue light in a chamber and those responding to the rising sun on their first day in the field exhibit similar bending, their underlying transcriptional responses are quite different. Our data also suggest the potential involvement of phytochrome-mediated signaling pathways in the initiation of solar tracking.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002344

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/