The auxin-responsive CsSPL9-CsGH3.4 module finely regulates auxin levels to suppress the development of adventitious roots in tea (Camellia sinensis).

The cutting technique is extensively used in tea breeding, with key emphasis on promoting the growth of adventitious roots (ARs). Despite its importance in tea cultivation, the mechanisms underlying AR development in tea remain unclear. In this study, we demonstrated the essential role of auxins in the initiation and progression of AR and established that the application of exogenous 1-naphthaleneacetic acid-enhanced AR formation in tissue-cultured seedlings and cuttings. Then, we found that the auxin-responsive transcription factor CsSPL9 acted as a negative regulator of AR development by reducing the levels of free indole-3-acetic acid (IAA) in tea plants. Furthermore, we identified CsGH3.4 as a downstream target of CsSPL9, which was activated by direct binding to its promoter. CsGH3.4 also inhibited AR development and maintained low levels of free IAA. Thus, these results revealed the inhibitory effect of the auxin-responsive CsSPL9-CsGH3.4 module on AR development by reducing free IAA levels in tea. These findings have significant theoretical and practical value for enhancing tea breeding practices.

Identification and Validation of the miR156 Family Involved in Drought Responses and Tolerance in Tea Plants (Camellia sinensis (L.) O. Kuntze).

The microRNA156 (miR156) family, one of the first miRNA families discovered in plants, plays various important roles in plant growth and resistance to various abiotic stresses. Previously, miR156s were shown to respond to drought stress, but miR156s in tea plants (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. Herein, we identify 47 mature sequences and 28 precursor sequences in tea plants. Our evolutionary analysis and multiple sequence alignment revealed that csn-miR156s were highly conserved during evolution and that the rates of the csn-miR156 members' evolution were different. The precursor sequences formed typical and stable stem-loop structures. The prediction of cis-acting elements in the CsMIR156s promoter region showed that the CsMIR156s had diverse cis-acting elements; of these, 12 CsMIR156s were found to be drought-responsive elements. The results of reverse transcription quantitative PCR (RT-qPCR) testing showed that csn-miR156 family members respond to drought and demonstrate different expression patterns under the conditions of drought stress. This suggests that csn-miR156 family members may be significantly involved in the response of tea plants to drought stress. Csn-miR156f-2-5p knockdown significantly reduced the Fv/Fm value and chlorophyll content and led to the accumulation of more-reactive oxygen species and proline compared with the control. The results of target gene prediction showed that csn-miR156f-2-5p targeted SQUAMOSA promoter binding protein-like (SPL) genes. Further analyses showed that CsSPL14 was targeted by csn-miR156f-2-5p, as confirmed through RT-qPCR, 5' RLM-RACE, and antisense oligonucleotide validation. Our results demonstrate that csn-miR156f-2-5p and CsSPL14 are involved in drought response and represent a new strategy for increasing drought tolerance via the breeding of tea plants.

Low-affinity SPL binding sites contribute to subgenome expression divergence in allohexaploid wheat.

Expression divergence caused by genetic variation and crosstalks among subgenomes of the allohexaploid bread wheat (Triticum aestivum. L., BBAADD) is hypothesized to increase its adaptability and/or plasticity. However, the molecular basis of expression divergence remains unclear. Squamosa promoter-binding protein-like (SPL) transcription factors are critical for a wide array of biological processes. In this study, we constructed expression regulatory networks by combining DAP-seq for 40 SPLs, ATAC-seq, and RNA-seq. Our findings indicate that a group of low-affinity SPL binding regions (SBRs) were targeted by diverse SPLs and caused different sequence preferences around the core GTAC motif. The SBRs including the low-affinity ones are evolutionarily conserved, enriched GWAS signals related to important agricultural traits. However, those SBRs are highly diversified among the cis-regulatory regions (CREs) of syntenic genes, with less than 8% SBRs coexisting in triad genes, suggesting that CRE variations are critical for subgenome differentiations. Knocking out of TaSPL7A/B/D and TaSPL15A/B/D subfamily further proved that both high- and low-affinity SBRs played critical roles in the differential expression of genes regulating tiller number and spike sizes. Our results have provided baseline data for downstream networks of SPLs and wheat improvements and revealed that CRE variations are critical sources for subgenome divergence in the allohexaploid wheat.

Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases.

Exploding seed pods evolved in the Arabidopsis relative Cardamine hirsuta via morphomechanical innovations that allow the storage and rapid release of elastic energy. Asymmetric lignin deposition within endocarpb cell walls is one such innovation that is required for explosive seed dispersal and evolved in association with the trait. However, the genetic control of this novel lignin pattern is unknown. Here, we identify three lignin-polymerizing laccases, LAC4, 11, and 17, that precisely colocalize with, and are redundantly required for, asymmetric lignification of endocarpb cells. By screening for C. hirsuta mutants with less lignified fruit valves, we found that loss of function of the transcription factor gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 7 (SPL7) caused a reduction in endocarpb cell-wall lignification and a consequent reduction in seed dispersal range. SPL7 is a conserved regulator of copper homeostasis and is both necessary and sufficient for copper to accumulate in the fruit. Laccases are copper-requiring enzymes. We discovered that laccase activity in endocarpb cell walls depends on the SPL7 pathway to acclimate to copper deficiency and provide sufficient copper for lignin polymerization. Hence, SPL7 links mineral nutrition to efficient dispersal of the next generation.

Conservation and Divergence of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Family between Wheat and Rice.

The SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family affects plant architecture, panicle structure, and grain development, representing key genes for crop improvements. The objective of the present study is to utilize the well characterized SPLs' functions in rice to facilitate the functional genomics of TaSPL genes. To achieve these goals, we combined several approaches, including genome-wide analysis of TaSPLs, comparative genomic analysis, expression profiling, and functional study of TaSPL3 in rice. We established the orthologous relationships of 56 TaSPL genes with the corresponding OsSPLs, laying a foundation for the comparison of known SPL functions between wheat and rice. Some TaSPLs exhibited different spatial-temporal expression patterns when compared to their rice orthologs, thus implicating functional divergence. TaSPL2/6/8/10 were identified to respond to different abiotic stresses through the combination of RNA-seq and qPCR expression analysis. Additionally, ectopic expression of TaSPL3 in rice promotes heading dates, affects leaf and stem development, and leads to smaller panicles and decreased yields per panicle. In conclusion, our work provides useful information toward cataloging of the functions of TaSPLs, emphasized the conservation and divergence between TaSPLs and OsSPLs, and identified the important SPL genes for wheat improvement.

PIL transcription factors directly interact with SPLs and repress tillering/branching in plants.

Tillering is an important parameter of plant architecture in cereal crops. In this study, we identified the PHYTOCHROME-INTERACTING FACTOR-LIKE (PIL) family transcription factors as new repressors of tillering in cereal crops. Using biochemical and genetic approaches, we explore the roles of TaPIL1 in regulating wheat plant architecture. We found that the PIL protein TaPIL1 controls tiller number in wheat. Overexpression of TaPIL1 reduces wheat tiller number; additionally, overexpression of TaPIL1-SUPERMAN repression domain increases wheat tiller number. Furthermore, we show that TaPIL1 activates the transcriptional expression of wheat TEOSINTE BRANCHED1 (TaTB1); moreover, TaPIL1 physically interacts with wheat SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (TaSPL)3/17, which are activators of TaTB1 transcription. In rice, overexpression and loss-of-function mutations of OsPIL11 reduce or increase tiller number by regulating the expression of OsTB1. In Arabidopsis, we demonstrate that PHYTOCHROME-INTERACTING FACTOR 4 interacts with SPL9 to inhibit shoot branching. This study reveals that PIL family transcription factors directly interact with SPLs and play an important role in repressing tillering/branching in plants.

Distinct Evolutionary Profiles and Functions of microRNA156 and microRNA529 in Land Plants.

MicroRNA156 (miR156) and miR529 have high sequence similarity and recognize overlapping sites in the same target genes, SQUAMOSA promoter binding protein-like (SPL or SBP box) genes, making it difficult to accurately distinguish their roles in regulatory networks that affect numerous biological functions. Here, we collected data about miR156 and miR529 family members from representative land plants and performed sequence comparisons, phylogenetic analysis, small RNA sequencing, and parallel analysis of RNA ends (PARE) analysis to dissect their evolutionary and functional differences. Although miR156 and miR529 are highly similar, there are differences in their mismatch-sensitive regions, which are essential for target recognition. In land plants, miR156 precursors are conserved mainly within the hairpin region, whereas miR529 precursors are conserved outside the hairpin region, including both the 5' and 3' arms. Phylogenetic analysis showed that MIR156 and MIR529 evolved independently, through divergent evolutionary patterns. The two genes also exhibit different expression patterns, with MIR529 preferentially expressed in reproductive tissues and MIR156 in other tissues. PARE analysis revealed that miR156 and miR529 possess specific targets in addition to common targets in maize, pointing to functional differences between them. Based on our findings, we developed a method for the rapid identification of miR529 and miR156 family members and uncovered the evolutionary divergence of these families, providing insights into their different regulatory roles in plant growth and development.

Ginsenosides in Panax genus and their biosynthesis.

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of Panax genus. They are accumulated in plant roots, stems, leaves, and flowers. The content and composition of ginsenosides are varied in different ginseng species, and in different parts of a certain plant. In this review, we summarized the representative saponins structures, their distributions and the contents in nearly 20 Panax species, and updated the biosynthetic pathways of ginsenosides focusing on enzymes responsible for structural diversified ginsenoside biosynthesis. We also emphasized the transcription factors in ginsenoside biosynthesis and non-coding RNAs in the growth of Panax genus plants, and highlighted the current three major biotechnological applications for ginsenosides production. This review covered advances in the past four decades, providing more clues for chemical discrimination and assessment on certain ginseng plants, new perspectives for rational evaluation and utilization of ginseng resource, and potential strategies for production of specific ginsenosides.

Glucose- and sucrose-signaling modules regulate the Arabidopsis juvenile-to-adult phase transition.

CINV1, converting sucrose into glucose and fructose, is a key entry of carbon into cellular metabolism, and HXK1 functions as a pivotal sensor for glucose. Exogenous sugars trigger the Arabidopsis juvenile-to-adult phase transition via a miR156A/SPL module. However, the endogenous factors that regulate this process remain unclear. In this study, we show that sucrose specifically induced the PAP1 transcription factor directly and positively controls CINV1 activity. Furthermore, we identify a glucose feed-forward loop (sucrose-CINV1-glucose-HXK1-miR156-SPL9-PAP1-CINV1-glucose) that controls CINV1 activity to convert sucrose into glucose signaling to dynamically control the juvenile-to-adult phase transition. Moreover, PAP1 directly binds to the SPL9 promoter, activating SPL9 expression and triggering the sucrose-signaling-mediated juvenile-to-adult phase transition. Therefore, a glucose-signaling feed-forward loop and a sucrose-signaling pathway synergistically regulate the Arabidopsis juvenile-to-adult phase transition. Collectively, we identify a molecular link between the major photosynthate sucrose, the entry point of carbon into cellular metabolism, and the plant juvenile-to-adult phase transition.

Genome-wide analysis of microRNA156 and its targets, the genes encoding SQUAMOSA promoter-binding protein-like (SPL) transcription factors, in the grass family Poaceae.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play an important role in post-transcriptional gene regulation in plants and animals by targeting messenger RNAs (mRNAs) for cleavage or repressing translation of specific mRNAs. The first miRNA identified in plants, miRNA156 (miR156), targets the SQUAMOSA promoter-binding protein-like (SPL) transcription factors, which play critical roles in plant phase transition, flower and plant architecture, and fruit development. We identified multiple copies of MIR156 and SPL in the rice, Brachypodium, sorghum, maize, and foxtail millet genomes. Sequence and chromosomal synteny analysis showed that both MIR156s and SPLs are conserved across species in the grass family. Analysis of expression data of the SPLs in eleven juvenile and adult rice tissues revealed that four non-miR156-targeted genes were highly expressed and three miR156-targeted genes were only slightly expressed in all tissues/developmental stages. The remaining SPLs were highly expressed in the juvenile stage, but their expression was lower in the adult stage. It has been proposed that under strong selective pressure, non-miR156-targeted mRNA may be able to re-structure to form a miRNA-responsive element. In our analysis, some non-miR156-targeted SPLs (SPL5/8/10) had gene structure and gene expression patterns similar to those of miR156-targeted genes, suggesting that they could diversify into miR156-targeted genes. DNA methylation profiles of SPLs and MIR156s in different rice tissues showed diverse methylation patterns, and hypomethylation of non-CG sites was observed in rice endosperm. Our findings suggested that MIR156s and SPLs had different origination and evolutionary mechanisms: the SPLs appear to have resulted from vertical evolution, whereas MIR156s appear to have resulted from strong evolutionary selection on mature sequences.

Genome-wide analysis of microRNA156 and its targets, the genes encoding SQUAMOSA promoter-binding protein-like (SPL) transcription factors, in the grass family Poaceae / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play an important role in post-transcriptional gene regulation in plants and animals by targeting messenger RNAs (mRNAs) for cleavage or repressing translation of specific mRNAs. The first miRNA identified in plants, miRNA156 (miR156), targets the SQUAMOSA promoter-binding protein-like (SPL) transcription factors, which play critical roles in plant phase transition, flower and plant architecture, and fruit development. We identified multiple copies of

The miR156/SPL module regulates apple salt stress tolerance by activating MdWRKY100 expression.

Salt stress dramatically impedes plant growth and development as well as crop yield. The apple production regions are reduced every year, because of the secondary salt damage by improper fertilization and irrigation. To expand the cultivation area of apple (Malus domestica) and select salt-resistant varieties, the mechanism of salt tolerance in apple is necessary to be clarified. The miR156/SPL regulatory module plays key roles in embryogenesis, morphogenesis, life cycle stage transformation, flower formation and other processes. However, its roles in the mechanisms of salt tolerance are unknown. In order to elucidate the mechanism of 156/SPL regulating salt stress in apple, we performed RLM-5' RACE and stable genetic transformation technology to verify that both mdm-MIR156a and MdSPL13 responded to salt stress in apple and that the latter was the target of the former. MIR156a overexpression weakened salt resistance in apple whereas MdSPL13 overexpression strengthened it. A total of 6094 differentially expressed genes relative to nontransgenic apple plants were found by RNA-Seq analysis of MdSPL13OE. Further verification indicated that MdSPL13 targeted the MdWRKY100 gene promoter. Moreover, MdWRKY100 overexpression enhanced salt tolerance in apple. Our results revealed that the miR156/SPL module regulates salt tolerance by up-regulating MdWRKY100 in apple. This study is the first to elucidate the mechanism underlying the miRNA network response to salt stress in apple and provides theoretical and empirical bases and genetic resources for the molecular breeding of salt tolerance in apple.

Gene regulatory networks controlled by FLOWERING LOCUS C that confer variation in seasonal flowering and life history.

Responses to environmental cues synchronize reproduction of higher plants to the changing seasons. The genetic basis of these responses has been intensively studied in the Brassicaceae. The MADS-domain transcription factor FLOWERING LOCUS C (FLC) plays a central role in the regulatory network that controls flowering of Arabidopsis thaliana in response to seasonal cues. FLC blocks flowering until its transcription is stably repressed by extended exposure to low temperatures in autumn or winter and, therefore, FLC activity is assumed to limit flowering to spring. Recent reviews describe the complex epigenetic mechanisms responsible for FLC repression in cold. We focus on the gene regulatory networks controlled by FLC and how they influence floral transition. Genome-wide approaches determined the in vivo target genes of FLC and identified those whose transcription changes during vernalization or in flc mutants. We describe how studying FLC targets such as FLOWERING LOCUS T, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15, and TARGET OF FLC AND SVP 1 can explain different flowering behaviours in response to vernalization and other environmental cues, and help define mechanisms by which FLC represses gene transcription. Elucidating the gene regulatory networks controlled by FLC provides access to the developmental and physiological mechanisms that regulate floral transition.

The trehalose 6-phosphate pathway impacts vegetative phase change in Arabidopsis thaliana.

The vegetative phase change marks the beginning of the adult phase in the life cycle of plants and is associated with a gradual decline in the microRNA miR156, in response to sucrose status. Trehalose 6-phosphate (T6P) is a sugar molecule with signaling function reporting the current sucrose state. To elucidate the role of T6P signaling in vegetative phase change, molecular, genetic, and metabolic analyses were performed using Arabidopsis thaliana loss-of-function lines in TREHALOSE PHOSPHATE SYNTHASE1 (TPS1), a gene coding for an enzyme that catalyzes the production of T6P. These lines show a significant delay in vegetative phase change, under both short and long day conditions. Induced expression of TPS1 complements this delay in the TPS1 knockout mutant (tps1-2 GVG::TPS1). Further analyses indicate that the T6P pathway promotes vegetative phase transition by suppressing miR156 expression and thereby modulating the levels of its target transcripts, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes. TPS1 knockdown plants, with a delayed vegetative phase change phenotype, accumulate significantly more sucrose than wild-type plants as a result of a feedback mechanism. In summary, we conclude that the T6P pathway forms an integral part of an endogenous mechanism that influences phase transitions dependent on the metabolic state.

The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa.

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Do Arabidopsis Squamosa promoter binding Protein-Like genes act together in plant acclimation to copper or zinc deficiency?

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)-dependent proteins, which includes enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu-deficient growth conditions, Squamosa promoter binding Protein-Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems, and it mediates a metabolic reorganization to economize on Cu. The transcription factor SPL7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by miRNA156 with roles in plant development. SPL7 shares extended regions of sequence homology with SPL1 and SPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with both the wild type and the spl7 single mutant under normal and Cu-deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL7, but provided no indication for important roles of SPL1 or SPL12 in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn-deficient wild type plants. Known Zn deficiency responses were unaltered in spl7, spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZnSOD activity is strongly downregulated in Zn-deficient A. thaliana, in association with an about 94% reduction in the abundance of the CSD2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL7 and not associated with an upregulation of MIR398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 of Chlamydomonas. In the future, resolving how the specificity of SPL protein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of different SPL family proteins in the regulation of either Cu deficiency responses or growth and development of land plants.

Expression Pattern of FT/TFL1 and miR156-Targeted SPL Genes Associated with Developmental Stages in Dendrobium catenatum.

Time to flower, a process either referring to juvenile-adult phase change or vegetative-reproductive transition, is strictly controlled by an intricate regulatory network involving at least both FT/TFL1 and the micro RNA (miR)156-regulated SPL family members. Despite substantial progresses recently achieved in Arabidopsis and other plant species, information regarding the involvement of these genes during orchid development and flowering competence is still limited. Dendrobium catenatum, a popular orchid species, exhibits a juvenile phase of at least three years. Here, through whole-genome mining and whole-family expression profiling, we analyzed the homologous genes of FT/TFL1, miR156, and SPL with special reference to the developmental stages. The FT/TFL1 family contains nine members; among them, DcHd3b transcribes abundantly in young and juvenile tissues but not in adult, contrasting with the low levels of others. We also found that mature miR156, encoded by a single locus, accumulated in large quantity in protocorms and declined by seedling development, coincident with an increase in transcripts of three of its targeted SPL members, namely DcSPL14, DcSPL7, and DcSPL18. Moreover, among the seven predicted miR156-targeted SPLs, only DcSPL3 was significantly expressed in adult plants and was associated with plant maturation. Our results might suggest that the juvenile phase change or maturation in this orchid plant likely involves both the repressive action of a TFL1-like pathway and the promotive effect from an SPL3-mediated mechanism.

Nitrate acts at the Arabidopsis thaliana shoot apical meristem to regulate flowering time.

Optimal timing of flowering, a major determinant for crop productivity, is controlled by environmental and endogenous cues. Nutrients are known to modify flowering time; however, our understanding of how nutrients interact with the known pathways, especially at the shoot apical meristem (SAM), is still incomplete. Given the negative side-effects of nitrogen fertilization, it is essential to understand its mode of action for sustainable crop production. We investigated how a moderate restriction by nitrate is integrated into the flowering network at the SAM, to which plants can adapt without stress symptoms. This condition delays flowering by decreasing expression of SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) at the SAM. Measurements of nitrate and the responses of nitrate-responsive genes suggest that nitrate functions as a signal at the SAM. The transcription factors NIN-LIKE PROTEIN 7 (NLP7) and NLP6, which act as master regulators of nitrate signaling by binding to nitrate-responsive elements (NREs), are expressed at the SAM and flowering is delayed in single and double mutants. Two upstream regulators of SOC1 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and SPL5) contain functional NREs in their promoters. Our results point at a tissue-specific, nitrate-mediated flowering time control in Arabidopsis thaliana.

Shade delays flowering in Medicago sativa.

Shade-intolerant plants respond to the decrease in the red (R) to far-red (FR) light ratio (R:FR) occurring under shade by elongating stems and petioles and by re-positioning leaves, in a race to outcompete neighbors for the sunlight resource. In some annual species, the shade avoidance syndrome (SAS) is accompanied by the early induction of flowering. Anticipated flowering is viewed as a strategy to set seeds before the resources become severely limiting. Little is known about the molecular mechanisms of SAS in perennial forage crops like alfalfa (Medicago sativa). To study SAS in alfalfa, we exposed alfalfa plants to simulated shade by supplementing with FR light. Low R:FR light produced a classical SAS, with increased internode and petiole lengths, but unexpectedly also with delayed flowering. To understand the molecular mechanisms involved in uncoupling SAS from early flowering, we used a transcriptomic approach. The SAS is likely to be mediated by increased expression of msPIF3 and msHB2 in low R:FR light. Constitutive expression of these genes in Arabidopsis led to SAS, including early flowering, strongly suggesting that their roles are conserved. Delayed flowering was likely to be mediated by the downregulation of msSPL3, which promotes flowering in both Arabidopsis and alfalfa. Shade-delayed flowering in alfalfa may be important to extend the vegetative phase under suboptimal light conditions, and thus assure the accumulation of reserves necessary to resume growth after the next season.

Constitutive Expression of Aechmea fasciata SPL14 (AfSPL14) Accelerates Flowering and Changes the Plant Architecture in Arabidopsis.

Variations in flowering time and plant architecture have a crucial impact on crop biomass and yield, as well as the aesthetic value of ornamental plants. Aechmea fasciata, a member of the Bromeliaceae family, is a bromeliad variety that is commonly cultivated worldwide. Here, we report the characterization of AfSPL14, a squamosa promoter binding protein-like gene in A. fasciata. AfSPL14 was predominantly expressed in the young vegetative organs of adult plants. The expression of AfSPL14 could be upregulated within 1 h by exogenous ethephon treatment. The constitutive expression of AfSPL14 in Arabidopsis thaliana caused early flowering and variations in plant architecture, including smaller rosette leaves and thicker and increased numbers of main inflorescences. Our findings suggest that AfSPL14 may help facilitate the molecular breeding of A. fasciata, other ornamental and edible bromeliads (e.g., pineapple), and even cereal crops.