Atmospheric nitrogen dioxide suppresses the activity of phytochrome interacting factor 4 to suppress hypocotyl elongation.

MAIN CONCLUSION: Ambient concentrations of atmospheric nitrogen dioxide (NO2) inhibit the binding of PIF4 to promoter regions of auxin pathway genes to suppress hypocotyl elongation in Arabidopsis. Ambient concentrations (10-50 ppb) of atmospheric nitrogen dioxide (NO2) positively regulate plant growth to the extent that organ size and shoot biomass can nearly double in various species, including Arabidopsis thaliana (Arabidopsis). However, the precise molecular mechanism underlying NO2-mediated processes in plants, and the involvement of specific molecules in these processes, remain unknown. We measured hypocotyl elongation and the transcript levels of PIF4, encoding a bHLH transcription factor, and its target genes in wild-type (WT) and various pif mutants grown in the presence or absence of 50 ppb NO2. Chromatin immunoprecipitation assays were performed to quantify binding of PIF4 to the promoter regions of its target genes. NO2 suppressed hypocotyl elongation in WT plants, but not in the pifq or pif4 mutants. NO2 suppressed the expression of target genes of PIF4, but did not affect the transcript level of the PIF4 gene itself or the level of PIF4 protein. NO2 inhibited the binding of PIF4 to the promoter regions of two of its target genes, SAUR46 and SAUR67. In conclusion, NO2 inhibits the binding of PIF4 to the promoter regions of genes involved in the auxin pathway to suppress hypocotyl elongation in Arabidopsis. Consequently, PIF4 emerges as a pivotal participant in this regulatory process. This study has further clarified the intricate regulatory mechanisms governing plant responses to environmental pollutants, thereby advancing our understanding of how plants adapt to changing atmospheric conditions.

A cytosol-tethered YHB variant of phytochrome B retains photomorphogenic signaling activity.

The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks therein. This necessitates changes in protein-protein interactions of phyB in the cytosol, about which little is presently known. Via introduction of a nucleus-excluding G767R mutation into the dominant, constitutively active phyBY276H (YHB) allele, we explore the functional consequences of expressing a cytosol-localized YHBG767R variant in transgenic Arabidopsis seedlings. We show that YHBG767R elicits selective constitutive photomorphogenic phenotypes in dark-grown phyABCDE null mutants, wild type and other phy-deficient genotypes. These responses include light-independent apical hook opening, cotyledon unfolding, seed germination and agravitropic hypocotyl growth with minimal suppression of hypocotyl elongation. Such phenotypes correlate with reduced PIF3 levels, which implicates cytosolic targeting of PIF3 turnover or PIF3 translational inhibition by YHBG767R. However, as expected for a cytoplasm-tethered phyB, YHBG767R elicits reduced light-mediated signaling activity compared with similarly expressed wild-type phyB in phyABCDE mutant backgrounds. YHBG767R also interferes with wild-type phyB light signaling, presumably by formation of cytosol-retained and/or otherwise inactivated heterodimers. Our results suggest that cytosolic interactions with PIFs play an important role in phyB signaling even under physiological conditions.

A Novel 10-Base Pair Deletion in the First Exon of GmHY2a Promotes Hypocotyl Elongation, Induces Early Maturation, and Impairs Photosynthetic Performance in Soybean.

Plants photoreceptors perceive changes in light quality and intensity and thereby regulate plant vegetative growth and reproductive development. By screening a γ irradiation-induced mutant library of the soybean (Glycine max) cultivar "Dongsheng 7", we identified Gmeny, a mutant with elongated nodes, yellowed leaves, decreased chlorophyll contents, altered photosynthetic performance, and early maturation. An analysis of bulked DNA and RNA data sampled from a population segregating for Gmeny, using the BVF-IGV pipeline established in our laboratory, identified a 10 bp deletion in the first exon of the candidate gene Glyma.02G304700. The causative mutation was verified by a variation analysis of over 500 genes in the candidate gene region and an association analysis, performed using two populations segregating for Gmeny. Glyma.02G304700 (GmHY2a) is a homolog of AtHY2a in Arabidopsis thaliana, which encodes a PΦB synthase involved in the biosynthesis of phytochrome. A transcriptome analysis of Gmeny using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed changes in multiple functional pathways, including photosynthesis, gibberellic acid (GA) signaling, and flowering time, which may explain the observed mutant phenotypes. Further studies on the function of GmHY2a and its homologs will help us to understand its profound regulatory effects on photosynthesis, photomorphogenesis, and flowering time.

NAC transcription factor ATAF1 negatively modulates the PIF-regulated hypocotyl elongation under a short-day photoperiod.

Day length modulates hypocotyl elongation in seedlings to optimize their overall fitness. Variations in cell growth-associated genes are regulated by several transcription factors. However, the specific transcription factors through which the plant clock increases plant fitness are still being elucidated. In this study, we identified the no apical meristem, Arabidopsis thaliana-activating factor (ATAF-1/2), and cup-shaped cotyledon (NAC) family transcription factor ATAF1 as a novel repressor of hypocotyl elongation under a short-day (SD) photoperiod. Variations in day length profoundly affected the transcriptional and protein levels of ATAF1. ATAF1-deficient mutant exhibited increased hypocotyl length and cell growth-promoting gene expression under SD conditions. Moreover, ATAF1 directly targeted and repressed the expression of the cycling Dof factor 1/5 (CDF1/5), two key transcription factors involved in hypocotyl elongation under SD conditions. Additionally, ATAF1 interacted with and negatively modulated the effects of phytochrome-interacting factor (PIF), thus inhibiting PIF-promoted gene expression and hypocotyl elongation. Taken together, our results revealed ATAF1-PIF as a crucial pair modulating the expression of key transcription factors to facilitate plant growth during day/night cycles under fluctuating light conditions.

miR394 modulates brassinosteroid signaling to regulate hypocotyl elongation in Arabidopsis.

The interplay between microRNAs (miRNAs) and phytohormones allows plants to integrate multiple internal and external signals to optimize their survival of different environmental conditions. Here, we report that miR394 and its target gene LEAF CURLING RESPONSIVENESS (LCR), which are transcriptionally responsive to BR, participate in BR signaling to regulate hypocotyl elongation in Arabidopsis thaliana. Phenotypic analysis of various transgenic and mutant lines revealed that miR394 negatively regulates BR signaling during hypocotyl elongation, whereas LCR positively regulates this process. Genetically, miR394 functions upstream of BRASSINOSTEROID INSENSITIVE2 (BIN2), BRASSINAZOLEs RESISTANT1 (BZR1), and BRI1-EMS-SUPPRESSOR1 (BES1), but interacts with BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1 SUPRESSOR PROTEIN (BSU1). RNA-sequencing analysis suggested that miR394 inhibits BR signaling through BIN2, as miR394 regulates a significant number of genes in common with BIN2. Additionally, miR394 increases the accumulation of BIN2 but decreases the accumulation of BZR1 and BES1, which are phosphorylated by BIN2. MiR394 also represses the transcription of PACLOBUTRAZOL RESISTANCE1/5/6 and EXPANSIN8, key genes that regulate hypocotyl elongation and are targets of BZR1/BES1. These findings reveal a new role for a miRNA in BR signaling in Arabidopsis.

Non-transcriptional regulatory activity of SMAX1 and SMXL2 mediates karrikin-regulated seedling response to red light in Arabidopsis.

Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways. The transcriptional repressor proteins SUPPRESSOR OF MAX2 1 (SMAX1), SMAX1-like2 (SMXL2), and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or by inhibiting the activities of transcription factors. In this study, we characterized the non-transcriptional regulatory activities of SMXL proteins in Arabidopsis. We discovered that SMAX1 and SMXL2 with mutations in their ethylene-response factor-associated amphiphilic repression (EAR) motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 mutant. SMAX1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR 4 (PIF4) and PIF5 to enhance their protein stability by interacting with phytochrome B (phyB) and suppressing the association of phyB with PIF4 and PIF5. The karrikin-responsive genes were then identified by treatment with GR24ent-5DS, a GR24 analog showing karrikin activity. Interestingly, INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) expression was repressed by GR24ent-5DS treatment in a PIF4- and PIF5-dependent and EAR-independent manner, whereas KARRIKIN UPREGULATED F-BOX 1 (KUF1) expression was induced in a PIF4- and PIF5-independent and EAR-dependent manner. Furthermore, the non-transcriptional regulatory activity of SMAX1, which is independent of the EAR motif, had a global effect on gene expression. Taken together, these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.

ADA2b acts to positively regulate blue light-mediated photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) act as blue light photoreceptors to regulate various plant physiological processes including photomorphogenesis and repair of DNA double strand breaks (DSBs). ADA2b is a conserved transcription co-activator that is involved in multiple plant developmental processes. It is known that ADA2b interacts with CRYs to mediate blue light-promoted DSBs repair. Whether ADA2b may participate in CRYs-mediated photomorphogenesis is unknown. Here we show that ADA2b acts to inhibit hypocotyl elongation and hypocotyl cell elongation in blue light. We found that the SWIRM domain-containing C-terminus mediates the blue light-dependent interaction of ADA2b with CRYs in blue light. Moreover, ADA2b and CRYs act to co-regulate the expression of hypocotyl elongation-related genes in blue light. Based on previous studies and these results, we propose that ADA2b plays dual functions in blue light-mediated DNA damage repair and photomorphogenesis.

B-Box transcription factor BBX28 requires CONSTITUTIVE PHOTOMORPHOGENESIS1 to induce shade-avoidance response in Arabidopsis thaliana.

Shade avoidance syndrome is an important adaptive strategy. Under shade, major transcriptional rearrangements underlie the reallocation of resources to elongate vegetative structures and redefine the plant architecture to compete for photosynthesis. BBX28 is a B-box transcription factor involved in seedling de-etiolation and flowering in Arabidopsis (Arabidopsis thaliana), but its function in shade-avoidance response is completely unknown. Here, we studied the function of BBX28 using two mutant and two transgenic lines of Arabidopsis exposed to white light and simulated shade conditions. We found that BBX28 promotes hypocotyl growth under shade through the phytochrome system by perceiving the reduction of red photons but not the reduction of photosynthetically active radiation or blue photons. We demonstrated that hypocotyl growth under shade is sustained by the protein accumulation of BBX28 in the nuclei in a CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1)-dependent manner at the end of the photoperiod. BBX28 up-regulates the expression of transcription factor- and auxin-related genes, thereby promoting hypocotyl growth under prolonged shade. Overall, our results suggest the role of BBX28 in COP1 signaling to sustain the shade-avoidance response and extend the well-known participation of other members of BBX transcription factors for fine-tuning plant growth under shade.

The phytochrome-interacting factor genes PIF1 and PIF4 are functionally diversified due to divergence of promoters and proteins.

Phytochrome-interacting factors (PIFs) are basic helix-loop-helix transcription factors that regulate light responses downstream of phytochromes. In Arabidopsis (Arabidopsis thaliana), 8 PIFs (PIF1-8) regulate light responses, either redundantly or distinctively. Distinctive roles of PIFs may be attributed to differences in mRNA expression patterns governed by promoters or variations in molecular activities of proteins. However, elements responsible for the functional diversification of PIFs have yet to be determined. Here, we investigated the role of promoters and proteins in the functional diversification of PIF1 and PIF4 by analyzing transgenic lines expressing promoter-swapped PIF1 and PIF4, as well as chimeric PIF1 and PIF4 proteins. For seed germination, PIF1 promoter played a major role, conferring dominance to PIF1 gene with a minor contribution from PIF1 protein. Conversely, for hypocotyl elongation under red light, PIF4 protein was the major element conferring dominance to PIF4 gene with the minor contribution from PIF4 promoter. In contrast, both PIF4 promoter and PIF4 protein were required for the dominant role of PIF4 in promoting hypocotyl elongation at high ambient temperatures. Together, our results support that the functional diversification of PIF1 and PIF4 genes resulted from contributions of both promoters and proteins, with their relative importance varying depending on specific light responses.

A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation.

The Xishuangbanna (XIS) cucumber (Cucumis sativus var. xishuangbannanesis) is a semiwild variety that has many distinct agronomic traits. Here, long reads generated by Nanopore sequencing technology helped assembling a high-quality genome (contig N50 = 8.7 Mb) of landrace XIS49. A total of 10,036 structural/sequence variations (SVs) were identified when comparing with Chinese Long (CL), and known SVs controlling spines, tubercles, and carpel number were confirmed in XIS49 genome. Two QTLs of hypocotyl elongation under low light, SH3.1 and SH6.1, were fine-mapped using introgression lines (donor parent, XIS49; recurrent parent, CL). SH3.1 encodes a red-light receptor Phytochrome B (PhyB, CsaV3_3G015190). A ∼4 kb region with large deletion and highly divergent regions (HDRs) were identified in the promoter of the PhyB gene in XIS49. Loss of function of this PhyB caused a super-long hypocotyl phenotype. SH6.1 encodes a CCCH-type zinc finger protein FRIGIDA-ESSENTIAL LIKE (FEL, CsaV3_6G050300). FEL negatively regulated hypocotyl elongation but it was transcriptionally suppressed by long terminal repeats retrotransposon insertion in CL cucumber. Mechanistically, FEL physically binds to the promoter of CONSTITUTIVE PHOTOMORPHOGENIC 1a (COP1a), regulating the expression of COP1a and the downstream hypocotyl elongation. These above results demonstrate the genetic mechanism of cucumber hypocotyl elongation under low light.

Arabidopsis hypocotyl growth in darkness requires the phosphorylation of a microtubule-associated protein.

Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de-etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho-signaling responders to light. Here, we reported a large-scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule-associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1-INTERACTING-PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine915 was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7-YFP and the phospho-mimetic CIP7S915D-YFP, but not the phospho-null CIP7S915A-YFP suggesting that the phosphorylated S915CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine915 of CIP7 unveils phospho-regulation of MT-based processes during skotomorphogenic hypocotyl growth.

JASMONATE ZIM-domain protein 3 regulates photomorphogenesis and thermomorphogenesis through inhibiting PIF4 in Arabidopsis.

Light and temperature are 2 major environmental factors that affect the growth and development of plants during their life cycle. Plants have evolved complex mechanisms to adapt to varying external environments. Here, we show that JASMONATE ZIM-domain protein 3 (JAZ3), a jasmonic acid signaling component, acts as a factor to integrate light and temperature in regulating seedling morphogenesis. JAZ3 overexpression transgenic lines display short hypocotyls under red, far-red, and blue light and warm temperature (28 °C) conditions compared to the wild type in Arabidopsis (Arabidopsis thaliana). We show that JAZ3 interacts with the transcription factor PHYTOCHROME-INTERACTING FACTOR4 (PIF4). Interestingly, JAZ3 spontaneously undergoes liquid-liquid phase separation (LLPS) in vitro and in vivo and promotes LLPS formation of PIF4. Moreover, transcriptomic analyses indicate that JAZ3 regulates the expression of genes involved in many biological processes, such as response to auxin, auxin-activated signaling pathway, regulation of growth, and response to red light. Finally, JAZ3 inhibits the transcriptional activation activity and binding ability of PIF4. Collectively, our study reveals a function and molecular mechanism of JAZ3 in regulating plant growth in response to environmental factors such as light and temperature.

CCaP1/CCaP2/CCaP3 interact with plasma membrane H+-ATPases and promote thermo-responsive growth by regulating cell wall modification in Arabidopsis.

Arabidopsis plants adapt to warm temperatures by promoting hypocotyl growth primarily through the basic helix-loop-helix transcription factor PIF4 and its downstream genes involved in auxin responses, which enhance cell division. In the current study, we discovered that cell wall-related calcium-binding protein 2 (CCaP2) and its paralogs CCaP1 and CCaP3 function as positive regulators of thermo-responsive hypocotyl growth by promoting cell elongation in Arabidopsis. Interestingly, mutations in CCaP1/CCaP2/CCaP3 do not affect the expression of PIF4-regulated classic downstream genes. However, they do noticeably reduce the expression of xyloglucan endotransglucosylase/hydrolase genes, which are involved in cell wall modification. We also found that CCaP1/CCaP2/CCaP3 are predominantly localized to the plasma membrane, where they interact with the plasma membrane H+-ATPases AHA1/AHA2. Furthermore, we observed that vanadate-sensitive H+-ATPase activity and cell wall pectin and hemicellulose contents are significantly increased in wild-type plants grown at warm temperatures compared with those grown at normal growth temperatures, but these changes are not evident in the ccap1-1 ccap2-1 ccap3-1 triple mutant. Overall, our findings demonstrate that CCaP1/CCaP2/CCaP3 play an important role in controlling thermo-responsive hypocotyl growth and provide new insights into the alternative pathway regulating hypocotyl growth at warm temperatures through cell wall modification mediated by CCaP1/CCaP2/CCaP3.

Light-sensitive short hypocotyl genes confer symbiotic nodule identity in the legume Medicago truncatula.

Legumes produce specialized root nodules that are distinct from lateral roots in morphology and function, with nodules intracellularly hosting nitrogen-fixing bacteria. We have previously shown that a lateral root program underpins nodule initiation, but there must be additional developmental regulators that confer nodule identity. Here, we show two members of the LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) transcription factor family, predominantly known to define shoot meristem complexity and organ boundaries, function as regulators of nodule organ identity. In parallel to the root initiation program, LSH1/LSH2 recruit a program into the root cortex that mediates the divergence into nodules, in particular with cell divisions in the mid-cortex. This includes regulation of auxin and cytokinin, promotion of NODULE ROOT1/2 and Nuclear Factor YA1, and suppression of the lateral root program. A principal outcome of LSH1/LSH2 function is the production of cells able to accommodate nitrogen-fixing bacteria, a key feature unique to nodules.

Chromatin attachment to the nuclear matrix represses hypocotyl elongation in Arabidopsis thaliana.

The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.

Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Mutation in shoot-to-root mobile transcription factor, ELONGATED HYPOCOTYL 5, leads to low nicotine levels in tobacco.

Tobacco remains one of the most commercially important crops due to the parasympathomimetic alkaloid nicotine used in cigarettes. Most genes involved in nicotine biosynthesis are expressed in root tissues; however, their light-dependent regulation has not been studied. Here, we identified the ELONGATED HYPOCOTYL 5 homolog, NtHY5, from Nicotiana tabacum and demonstrated that NtHY5 could complement the Arabidopsis thaliana hy5 mutant at molecular, morphological and biochemical levels. We report the development of CRISPR/Cas9-based knockout mutant plants of tobacco, NtHY5CR, and show down-regulation of the nicotine and phenylpropanoid pathway genes leading to a significant reduction in nicotine and flavonol content, whereas NtHY5 overexpression (NtHY5OX) plants show the opposite effect. Grafting experiments using wild-type, NtHY5CR, and NtHY5OX indicated that NtHY5 moves from shoot-to-root to regulate nicotine biosynthesis in the root tissue. Shoot HY5, directly or through enhancing expression of the root HY5, promotes nicotine biosynthesis by binding to light-responsive G-boxes present in the NtPMT, NtQPT and NtODC promoters. We conclude that the mobility of HY5 from shoot-to-root regulates light-dependent nicotine biosynthesis. The CRISPR/Cas9-based mutants developed, in this study; with low nicotine accumulation in leaves could help people to overcome their nicotine addiction and the risk of death.

Air channels create a directional light signal to regulate hypocotyl phototropism.

In plants, light direction is perceived by the phototropin photoreceptors, which trigger directional growth responses known as phototropism. The formation of a phototropin activation gradient across a photosensitive organ initiates this response. However, the optical tissue properties that functionally contribute to phototropism remain unclear. In this work, we show that intercellular air channels limit light transmittance through various organs in several species. Air channels enhance light scattering in Arabidopsis hypocotyls, thereby steepening the light gradient. This is required for an efficient phototropic response in Arabidopsis and Brassica. We identified an embryonically expressed ABC transporter required for the presence of air channels in seedlings and a structure surrounding them. Our work provides insights into intercellular air space development or maintenance and identifies a mechanism of directional light sensing in plants.

Physiological Analysis and Genetic Mapping of Short Hypocotyl Trait in Brassica napus L.

Hypocotyl length is a botanical trait that affects the cold tolerance of Brassica napus L. (B. napus). In this study, we constructed an F2 segregating population using the cold-resistant short hypocotyl variety '16VHNTS158' and the cold-sensitive long hypocotyl variety 'Tianyou 2288' as the parents, and BSA-seq was employed to identify candidate genes for hypocotyl length in B. napus. The results of parental differences showed that the average hypocotyl lengths of '16VHNTS158' and 'Tianyou 2288' were 0.41 cm and 0.77 cm at the 5~6 leaf stage, respectively, after different low-temperature treatments, and '16VHNTS158' exhibited lower relative ion leakage rates compared to 'Tianyou 2288'. The contents of indole acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) in hypocotyls of '16VHNTS158' and 'Tianyou 2288' increased with decreasing temperatures, but the IAA and GA contents were significantly higher than those of 'Tianyou 2288', and the BR content was lower than that of 'Tianyou 2288'. The genetic analysis results indicate that the genetic model for hypocotyl length follows the 2MG-A model. By using SSR molecular markers, a QTL locus associated with hypocotyl length was identified on chromosome C04. The additive effect value of this locus was 0.025, and it accounted for 2.5% of the phenotypic variation. BSA-Seq further localized the major effect QTL locus on chromosome C04, associating it with 41 genomic regions. The total length of this region was 1.06 Mb. Within this region, a total of 20 non-synonymous mutation genes were identified between the parents, and 26 non-synonymous mutation genes were found within the pooled samples. In the reference genome of B. napus, this region was annotated with 24 candidate genes. These annotated genes are predominantly enriched in four pathways: DNA replication, nucleotide excision repair, plant hormone signal transduction, and mismatch repair. The findings of this study provide a theoretical basis for cloning genes related to hypocotyl length in winter rapeseed and their utilization in breeding.