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2024 =>
Adaptations to high latitude photoperiods have been under positive selection during the domestication of many short-day (SD) flowering crops. Photoperiod insensitivity (auto-flowering) in drug-type Cannabis sativa circumvents the need for SD flowering requirements making outdoor cultivation in...
www.biorxiv.org
Adaptations to high latitude photoperiods have been under positive selection during the domestication of many short-day (SD) flowering crops. Photoperiod insensitivity (auto-flowering) in drug-type Cannabis sativa circumvents the need for SD flowering requirements making outdoor cultivation in...
www.biorxiv.org
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Figure 3.
Mean expression levels of
CsHd3a and
CsPRR37 mRNA throughout development. Gray areas represent days under SD (12h:12h), while white areas indicate days under LD (18h:6h). Biological replicates were a minimum of N = 3, maximum of N = 4.
(A) CsHd3a expression in auto-flowering plants.
(B) CsHd3a expression in photoperiod sensitive plants.
(C) CsPRR37 expression. Black line indicates photoperiod sensitive plants and red dashed line auto-flowering plants.
"allele" normal i AF
Figure 4.
CsPRR37 mRNA transcipt variants identified in photoperiod sensitive and auto-flowering plants.
(A) Functional transcript identified in photoperiod sensitive plants. Position and sequence of cryptic splice sites used in transcripts isolated from auto-flowering plants are labeled on exon 2.
(B-F) Transcript variants identified in auto-flowering plants. Position of G to T mutation knocking out the donor splice site after exon 2 is depicted in B and is present in all auto-flowering transcript variants. Variant abundence in auto-flowering plants is labeled on the right side of each transcript (N = 27). Introns are shown as solid lines and exons as boxes. Orange boxes indicate in frame exons and beige boxes as frame shifted exons. Red and blue boxes indicate positions of in frame PR domain and CCT domain respectively.
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LD clock in auto-flowering Cannabis is disrupted
We next investigated whether the splice mutation identified in
CsPRR7 could disrupt the circadian clock within auto-flowering cannabis plants thereby effecting flowering time. Pseudoresponse regulators are important components of the circadian clock and are characterized by an oscillating expression pattern peaking at specific times of the light-dark cycle. To determine if
CsPRR37 exhibits the expected oscillating expression pattern, RT-qPCR was used to measure
CsPRR37 gene expression every 3 hours over a 24h period. Sampling was conducted on both photoperiod sensitive and auto-flowering genotypes grown under LD (18h light, 6h dark).
CsPRR37 exhibited a bi-phasic expression with a midday peak at Zeitgeber Time (ZT) 11 and nighttime peak between ZT 20 and ZT 23 (
Figure 5a). The nighttime peak was approximately twice that of the midday peak.
The expression profiles between auto-flowering and photoperiod sensitive CsPRR37 only differed in the amplitude of their midday peak where auto-flowering CsPRR37 showed a 50% reduction in expression compared to photoperiod CsPRR37. We hypothesized that in addition to the midday peak differences in CsPRR37 transcript levels, its predicted truncated protein products should disrupt the expression of other clock components, in particular, those directly regulated by CsPRR37.
Adaptation to Higher Latitudes may be Attributed to CsPRR37 Variation
Photoperiod insensitivity is a common trait observed in SD crops domesticated in higher latitudes. Intensive artificial selection in rice has allowed its adaptation to latitudes as high as 53°N. Rice grown at these latitudes flower extremely early with weak or no photoperiod sensitivity (
Wei et al., 2008;
Fujino and Sekiguchi 2005). Crosses between late flowering
indica cultivars with early flowering
japonica have identified many distinct heading date QTLs contributing to a diverse range of flowering times (
Yoo et al., 2007;
Uga et al., 2007;
Doi et al., 2004;
Monna et al., 2002). A major effect QTL
Hd2 was identified as
OsPRR37 (
Murakami et al., 2005;
Koo et al., 2013).
Koo et al. (2013) showed that natural variation in
OsPRR37 contributed to the expansion of rice cultivation in northern latitudes, where cultivars grown at the most northern regions contained loss-of-function alleles.
Recently,
Chen et al., (2022) collected both natural and domesticated accession of cannabis across China. Flowering time experiments showed that wild collections flowered under LD. All natural accessions except for two, were collected in northern China at latitudes ranging from 41.50 – 50.16°N. The two which were not from northern China were collected in northern Yunnan and Xizang province, close to the Tibetan plateau where cannabis is proposed to have originated (
McPartland et al., 2019). Gene expression analysis of florigen
CsHd3a showed strong upregulation under LD in wild accessions, similar to our observations in
Figure 3a. Furthermore, an analysis of nucleotide diversity between domesticated photoperiod sensitive and wild accessions identified genes under positive selection, of which
CsPRR37 was significant. Like rice, natural variation in
CsPRR37 may be involved in cannabis adaptation to varying latitudes by regulating flowering time, with loss-of-function alleles present in the highest latitude regions. A detailed analysis of
CsPRR37 in wild and domesticate genotypes adapted to varying latitudes will be required to confirm this hypothesis. Currently, sequence data from auto-flowering wild accession collected by
Chen et al., (2022) are under embargo, however, wild accession collected by
Ren et al., (2021) overlap geographical regions. While flowering time was not analyzed in this study, the search for
CsPRR37 mutation(s) within these accessions is underway.
CsPRR37 may repress CsHd3a, preventing flowering under LD
Photoperiod insensitive genotypes of cannabis were found to contain a splice site mutation in
CsPRR37. CsPRR37 is homologous to other PRR family genes within the PRR3/7 clade (
Hotta et al., 2022), many of which have been confirmed as
bona fide timekeepers such as
PRR7 in
Arabidopsis, and
PRR37 in rice.
CsPRR37 contains both PR and CCT domains which are signature features of pseudo-response regulators. The CCT domain is involved in nuclear localization and DNA binding (
Tiwari et al., 2010;
Robson et al., 2001). The PR domain is involved in forming heterodimers (
Yuan et al., 2021). Sequencing of
CsPRR37 transcripts from auto-flowing genotypes identified 5 variants, 3 of which had early stop codons and lacked a CCT domain. The other two transcript variants had deletions in the PR domain and an apparently functional CCT domain. These results suggest that the
CsPRR37 splice site mutation identified in auto-flowering plants likely causes a severe loss of function in the CsPRR37 protein due to a combination of lower gene expression and disrupted PR and CCT domains.
In
Arabidopsis, PRR7 forms a regulatory feedback loop repressing
CCA1 and
LHY expression in conjunction with
PRR9 and
PRR5 (
Nakamichi et al., 2010;
Farre et al., 2005). Double and triple PRR mutants involving non-functional PRR7 has been shown to disrupt
CCA1 and
LHY circadian rhythm effecting flowering time (
Nakamichi et al., 2007). Under both LD and SD conditions no differences in were observed in
CsLHY regulation between auto-flowering and photoperiod sensitive genotypes. This may be explained by the semi-redundant function of PRR genes, as observed in
Arabidopsis, but doesn’t explain the early flowering phenotype of auto-flowering cannabis. Alternatively,
CsPRR37 may have a more direct function in regulating flowering as it does in other SD flowering crops. In rice, OsPRR37 delays heading date by repressing
Hd3a under LDs independent of key photoperiod regulators
Hd1 (a CONSTANS ortholog) and
Ehd1 (
Koo et al., 2013; Yano et al., 2004).
A similar regulation in cannabis would explain auto-flowering where a non-functional CsPRR37 would fail to repress CsHd3a under LD. Indeed, CsHd3a expression is upregulated in auto-flowering plants in LD. It is interesting to note that the FT homologue we analyzed has a closer homology to rice Hd3a, which is a SD specific florigen as opposed to rice RFT1, a LD specific florigen.
Circadian Rhythms are Drastically Altered between SD and LD Photoperiods
Cannabis circadian clock genes were sequentially expressed during SD photoperiod between ZT 0 (lights on) and ZT 12 (lights off). Peak expression of clock genes occurred approximately every 3 hours in the following order:
CsLHY,
CsPRR37,
CsPRR95,
CsPRR3, and
CsPRR1/TOC1. This same pattern has been observed in the circadian clock genes of both
Arabidopsis and Rice, highlighting its conservation in higher plants (
Murakami et al., 2003;
Matsushika et al., 2000). Unexpectedly, this pattern deviated drastically when cannabis was grown under LD (18h light and 6h dark). Under this photoperiod
CsTOC1,
CsPRR95, and
CsPRR3 all have overlapping oscillations with peak expression at ZT 5. Most striking was the inverted oscillation peaks observed in
CsLHY and CsPRR1/TOC1. In
Arabidopsis, the regulatory feedback loop created by morning expressed gene
LHY (and homologous
CCA1) and evening expressed
TOC1 composes the core oscillator of the circadian clock (
Alabadi et al., 2001). This feedback loop appears to be conserved in other plant species (
Wang et al., 2011;
Murakami et al., 2007). In LD entrained cannabis,
CsLHY peak oscillation occurred at night (ZT 20), while
CsTOC/PRR1 expression peaked in the subjective morning at ZT 5.
It should be noted that an 18h:6h photoperiod is highly unnatural for cannabis and was chosen because of its use in the commercial production of drug-type cannabis. Therefore, the atypical oscillations observed under 18h:6h photoperiod may be the results of the circadian clock operating under a longer daylength than it evolved to do so.
CsPRR37 Biphasic Expression may be Light Dependent
Except for the phase advance observed in auto-flowering CsPRR1/TOC1, circadian rhythms between auto-flowering and photoperiod sensitive plants did not drastically differ under SD. During LD, both auto-flowering
CsPRR95,
CsPRR3, and
CsGI oscillations were altered. While not statistically significant,
CsPRR1/TOC1 peak expression was attenuated in auto-flowering plants under LD as well. The increased perturbation of circadian clock genes in auto-flowering plants in LD compared to SD may allude to a LD specific function of
CsPRR37. Interestingly, the daytime peak of
CsPRR37 seemed to fill a gap in the circadian clock, being the only gene upregulated between ZT 9 and ZT 14. Moreso,
CsPRR37 was the only circadian clock gene analyzed in this study that showed a biphasic expression pattern observed only under LD.
CsPRR37 expression showed peaks at ZT 11 (daytime) and ZT 20 - 23 (nighttime). A biphasic expression pattern has been reported in Sorghum, with
SbPRR37 similarly being expressed at ZT 3 and ZT 15 under LD photoperiods (
Murphy et al., 2011). Under SD,
SbPRR37 loses its evening peak. Constant light (LL) experiments to assess the free-running period of
SbPRR37 found that
SbPRR37 expression is light dependent, with both morning and evening peaks present under LD and LL, while SD and constant dark (DD) conditions yielding only the morning peak.
Murphy et al., (2011) demonstrated that
SbPRR37 is a central repressor in the flowering regulatory pathway in response to photoperiod, with both morning and evening peaks needed to sufficiently repress expression of
SbFT. Given the similarities in
CsPRR37 expression, LL and DD experiments are currently being conducted to assess the light dependency of
CsPRR37. These experiments will additionally be used to better understand the free running periods of circadian clock genes analyzed in this study, and how integral
CsPRR37 is to the clock’s proper function.
Conclusion
The large introgression carried in our diverse panel of auto-flowering genotypes (
Figure 1) is likely shared with most auto-flower accessions in cannabis and explained by the severely reduced recombination observed between photoperiod sensitive and auto-flowering haplotypes. Currently available auto-flowering genotypes are known to have lower cannabinoid concentrations and poor flower quality than photoperiod sensitive cannabis, likely due to other QTLs on this large introgression. Knowing the causal mutation, which we believe is in
CsPRR37, enables large scale screens for rare recombinants, as we have demonstrated, which we expect will break this linkage drag and facilitate the breeding of auto-flowering cultivars with competitive quality to photoperiod sensitive ones. Advancements in auto-flowering genetics will help transition the drug-type cannabis industry from clonally propagated indoor grown production to outdoor seed grown cultivars adapted to a diverse range of bioregions.
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