Sequential activation of strigolactone and salicylate biosynthesis promotes leaf senescence.

Leaf senescence is a complex process strictly regulated by various external and endogenous factors. However, the key signaling pathway mediating leaf senescence remains unknown. Here, we show that Arabidopsis SPX1/2 negatively regulate leaf senescence genetically downstream of the strigolactone (SL) pathway. We demonstrate that the SL receptor AtD14 and MAX2 mediate the age-dependent degradation of SPX1/2. Intriguingly, we uncover an age-dependent accumulation of SLs in leaves via transcriptional activation of SL biosynthetic genes by the transcription factors (TFs) SPL9/15. Furthermore, we reveal that SPX1/2 interact with the WRKY75 subclade TFs to inhibit their DNA-binding ability and thus repress transcriptional activation of salicylic acid (SA) biosynthetic gene SA Induction-Deficient 2, gating the age-dependent SA accumulation in leaves at the leaf senescence onset stage. Collectively, our new findings reveal a signaling pathway mediating sequential activation of SL and salicylate biosynthesis for the onset of leaf senescence in Arabidopsis.

The transcription factor OsSPL9 endows rice with copper deficiency resilience.

Copper (Cu) is a crucial micronutrient essential for the growth and development of plants. Rice exhibits remarkable resistance to Cu deficiency, but the underlying molecular mechanisms are not well understood. In this study, we reveal that the plant's ability to withstand Cu deficiency is orchestrated by a transcription factor known as OsSPL9. We have demonstrated that OsSPL9 functions as a central regulator of Cu homeostasis. Disrupting OsSPL9 through knockout significantly reduced the plant's tolerance to Cu deficiency. As a result, the spl9 mutants exhibited reduced Cu accumulation in their shoots when compared with wild-type plants. This reduction was linked to a disruption in the transport of Cu from older leaves to younger ones. Furthermore, we show that OsSPL9 directly bound to GTAC motifs in the promoters of key genes involved in Cu uptake and transport, as well as Cu-miRNAs, and enhanced their transcription under Cu-deficient conditions. Overall, our findings shed light on the molecular basis of rice resilience to Cu deficiency stress and place the transcription factor OsSPL9 as a master regulator of this response.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 13 (SLP13) together with SPL9 redundantly regulates wax biosynthesis under drought stress.

Wax biosynthesis is closely controlled by many regulators under different environmental conditions. We have previously shown that the module miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)-DEWAX is involved in the diurnal regulation of wax production; however, it was not determined whether other SPLs are also involved in wax synthesis. Here, we report that SPL13 also regulates drought-induced wax production, by directly and indirectly affecting the expression of the two wax biosynthesis genes ECERIFERUM1 (CER1) and CER4, respectively. In addition, we show that SPL13 together with SPL9 redundantly regulates wax accumulation under both normal and drought stress conditions, and that simultaneous mutation of both genes additively increases cuticle permeability and decreases drought tolerance. However, in contrast to SPL9, SPL13 does not seem to participate in the DEWAX-mediated diurnal regulation of wax production.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 and 13 repress BLADE-ON-PETIOLE 1 and 2 directly to promote adult leaf morphology in Arabidopsis.

The juvenile-to-adult phase transition during vegetative development is a critical decision point in a plant's life cycle. This transition is mediated by a decline in levels of miR156/157 and an increase in the activities of its direct targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) proteins. In Arabidopsis, the juvenile-to-adult transition is characterized by an increase in the length to width ratio of the leaf blade (a change in the distal region of a leaf), but what mediates this change in lamina shape is not known. Here, we show that ectopic expression of SPL9 and SPL13 produces enlarged and elongated leaves, resembling leaves from the blade-on-petiole1 (bop1) bop2 double mutant. The expression of BOP1/BOP2 is down-regulated in successive leaves, correlating with the amount of miR156 and antagonistic to the expression of SPL9 and SPL13 in leaves. SPL9 and SPL13 bind to the promoters of BOP1/BOP2 directly to repress their expression, resulting in delayed establishment of proliferative regions in leaves, which promotes more blade outgrowth (the distal region of a leaf) and suppresses petiole development (the proximal region of a leaf). Our results reveal a mechanism for leaf development along the proximal-distal axis, a heteroblastic character between juvenile leaves and adult leaves.

SQUINT Positively Regulates Resistance to the Pathogen Botrytis cinerea via miR156-SPL9 Module in Arabidopsis.

SQUINT (SQN) regulates plant maturation by promoting the activity of miR156, which functions primarily in the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) module regulating plant growth and development. Here, we show that SQN acts in the jasmonate (JA) pathway, a major signaling pathway regulating plant responses to insect herbivory and pathogen infection. Arabidopsis thaliana sqn mutants showed elevated sensitivity to the necrotrophic fungus Botrytis cinerea compared with wild type. However, SQN is not involved in the early pattern-triggered immunity response often triggered by fungal attack. Rather, SQN positively regulates the JA pathway, as sqn loss-of-function mutants treated with B. cinerea showed reduced JA accumulation, JA response and sensitivity to JA. Furthermore, the miR156-SPL9 module regulates plant resistance to B. cinerea: mir156 mutant, and SPL9 overexpression plants displayed elevated sensitivity to B. cinerea. Moreover, constitutively expressing miR156a or reducing SPL9 expression in the sqn-1 mutant restored the sensitivity of Arabidopsis to B. cinerea and JA responses. These results suggest that SQN positively modulates plant resistance to B. cinerea through the JA pathway, and the miR156-SPL9 module functions as a bridge between SQN and JA to mediate plant resistance to this pathogen.

SPL9 mediates freezing tolerance by directly regulating the expression of CBF2 in Arabidopsis thaliana.

BACKGROUND: Freezing stress inhibits plant development and causes significant damage to plants. Plants therefore have evolved a large amount of sophisticated mechanisms to counteract freezing stress by adjusting their growth and development correspondingly. Plant ontogenetic defense against drought, high salt, and heat stresses, has been extensively studied. However, whether the freezing tolerance is associated with ontogenetic development and how the freezing signals are delivered remain unclear. RESULTS: In this study, we found that the freezing tolerance was increased with plant age at the vegetative stage. The expressions of microRNA156 (miR156) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), playing roles in regulation of ontogenetic development, were induced by cold stress. Overexpression of SPL9 (rSPL9) promoted the expression of C-REPEAT BINDING FACTOR 2 (CBF2) and hereafter enhanced the freezing tolerance. Genetic analysis indicated that the effect of rSPL9 on freezing tolerance is partially restored by cbf2 mutant. Further analysis confirmed that SPL9 directly binds to the promoter of CBF2 to activate the expression of CBF2, and thereafter increased the freezing tolerance. CONCLUSIONS: Therefore, our study uncovers a new role of SPL9 in fine-tuning CBF2 expression and thus mediating freezing tolerance in plants, and implies a role of miR156-SPL pathway in balancing the vegetative development and freezing response in Arabidopsis.

Molecular identification and functional verification of SPL9 and SPL15 of Lilium.

The transformation of plants from juveniles to adults is a key process in plant growth and development, and the main regulatory factors are miR156 and SQUAMOSA promoter binding protein-like (SPL) transcription factors. Lilium is an ornamental bulb, but it has a long maturation time. In this experiment, Lilium bulbs were subjected to a temperature treatment of 15 °C for 4 weeks to initiate vegetative phase change. Transmission electron microscopy indicated the cell wall of bud core tissue undergoing vegetative phase change became thinner, the starch grains were reduced, and the growth of the juvenile stage was accelerated. The key transcription factors LbrSPL9 and LbrSPL15 were cloned, and the phylogenetic analysis showed they possessed high homology with other plant SPLs. Subcellular localization and transcription activation experiments confirmed LbrSPL9 and LbrSPL15 were mainly located in the nucleus and exhibited transcriptional activity. The results of in situ hybridization showed the expression levels of LbrSPL9 and LbrSPL15 were increased after temperature change treatment. The functional verification experiment of the transgenic plants confirmed that the overexpression of LbrSPL9 and LbrSPL15 could shorten maturation time. These findings help elucidate the regulatory mechanisms of phase transition in Lilium and provide a reference for breeding research in other bulbous flowers.

BZR1 Physically Interacts with SPL9 to Regulate the Vegetative Phase Change and Cell Elongation in Arabidopsis.

As sessile organisms, the precise development phase transitions are very important for the success of plant adaptability, survival and reproduction. The transition from juvenile to the adult phase-referred to as the vegetative phase change-is significantly influenced by numbers of endogenous and environmental signals. Here, we showed that brassinosteroid (BR), a major growth-promoting steroid hormone, positively regulates the vegetative phase change in Arabidopsis thaliana. The BR-deficient mutant det2-1 and BR-insensitive mutant bri1-301 displayed the increased ratio of leaf width to length and reduced blade base angle. The plant specific transcription factors SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) are key masters for the vegetative phase transition in plants. The expression levels of SPL9, SPL10 and SPL15 were significantly induced by BR treatment, but reduced in bri1-116 mutant compared to wild-type plants. The gain-of-function pSPL9:rSPL9 transgenic plants displayed the BR hypersensitivity on hypocotyl elongation and partially suppressed the delayed vegetative phase change of det2-1 and bri1-301. Furthermore, we showed that BRASSINAZOLE-RESISTANT 1 (BZR1), the master transcription factor of BR signaling pathway, interacted with SPL9 to cooperatively regulate the expression of downstream genes. Our findings reveal an important role for BRs in promoting vegetative phase transition through regulating the activity of SPL9 at transcriptional and post-transcriptional levels.

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa.

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions.

The nuclear localization signal is required for the function of squamosa promoter binding protein-like gene 9 to promote vegetative phase change in Arabidopsis.

KEY MESSAGE: A mutation in the nuclear localization signal of squamosa promoter binding like-protein 9 (SPL9) delays vegetative phase change by disrupting its nuclear localization. The juvenile-to-adult phase transition is a critical developmental process in plant development, and it is regulated by a decrease in miR156/157 and a corresponding increase in their targets, squamosa promoter binding protein-like (SPL) genes. SPL proteins contain a conserved SBP domain with putative nuclear localization signals (NLSs) at their C-terminals. Some SPLs promote vegetative phase change by promoting miR172 expression, but the function of nuclear localization signals in those SPLs remains unknown. Here, we identified a loss-of-function mutant, which we named del6, with delayed vegetative phase change phenotypes in a forward genetic screen. Map-based cloning, the whole genome resequencing, and allelic complementation test demonstrate that a G-to-A substitution in the SPL9 gene is responsible for the delayed vegetative phase change phenotypes. In del6, the mutation causes a substitution of the glutamine (Gln) for the conserved basic amino acid arginine (Arg) in the NLS of the SBP domain, and disrupts the normal nuclear localization and function of SPL9. Therefore, our work demonstrates that the NLSs in the SBP domain of SPL9 are indispensable for its nuclear localization and normal function in Arabidopsis.

Study on Expression Modes and Cleavage Role of miR156b/c/d and its Target Gene Vv-SPL9 During the Whole Growth Stage of Grapevine.

miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine "Takatsuma" was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3'- and 5'-ends via an integrated approach of 5'-RLM-RACE (RNA ligase-mediated 5'-rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3'-rapid amplification of cDNA ends), and qRT-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal-spatial-specific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvi-miR156b/c/d complementary to Vv-SPL9 in "Takatsuma" grapevine. We also identified the temporal-spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine.

Age-associated growth control modifies leaf proximodistal symmetry and enabled leaf shape diversification.

Biological shape diversity is often manifested in modulation of organ symmetry and modification of the patterned elaboration of repeated shape elements.1,2,3,4,5 Whether and how these two aspects of shape determination are coordinately regulated is unclear.5,6,7 Plant leaves provide an attractive system to investigate this problem, because they often show asymmetries along the proximodistal (PD) axis of their blades, along which they can also produce repeated marginal outgrowths such as serrations or leaflets.1 One aspect of leaf shape diversity is heteroblasty, where the leaf form in a single genotype is modified with progressive plant age.8,9,10,11 In Arabidopsis thaliana, a plant with simple leaves, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) controls heteroblasty by activating CyclinD3 expression, thereby sustaining proliferative growth and retarding differentiation in adult leaves.12,13 However, the precise significance of SPL9 action for leaf symmetry and marginal patterning is unknown. By combining genetics, quantitative shape analyses, and time-lapse imaging, we show that PD symmetry of the leaf blade in A. thaliana decreases in response to an age-dependent SPL9 expression gradient, and that SPL9 action coordinately regulates the distribution and shape of marginal serrations and overall leaf form. Using comparative analyses, we demonstrate that heteroblastic growth reprogramming in Cardamine hirsuta, a complex-leafed relative of A. thaliana, also involves prolonging the duration of cell proliferation and delaying differentiation. We further provide evidence that SPL9 enables species-specific action of homeobox genes that promote leaf complexity. In conclusion, we identified an age-dependent layer of organ PD growth regulation that modulates leaf symmetry and has enabled leaf shape diversification.

Cell-cycle-linked growth reprogramming encodes developmental time into leaf morphogenesis.

How is time encoded into organ growth and morphogenesis? We address this question by investigating heteroblasty, where leaf development and form are modified with progressing plant age. By combining morphometric analyses, fate-mapping through live-imaging, computational analyses, and genetics, we identify age-dependent changes in cell-cycle-associated growth and histogenesis that underpin leaf heteroblasty. We show that in juvenile leaves, cell proliferation competence is rapidly released in a "proliferation burst" coupled with fast growth, whereas in adult leaves, proliferative growth is sustained for longer and at a slower rate. These effects are mediated by the SPL9 transcription factor in response to inputs from both shoot age and individual leaf maturation along the proximodistal axis. SPL9 acts by activating CyclinD3 family genes, which are sufficient to bypass the requirement for SPL9 in the control of leaf shape and in heteroblastic reprogramming of cellular growth. In conclusion, we have identified a mechanism that bridges across cell, tissue, and whole-organism scales by linking cell-cycle-associated growth control to age-dependent changes in organ geometry.

TRY intron2 determined its expression in inflorescence activated by SPL9 and MADS-box genes in Arabidopsis.

Plant trichomes are specialized epidermal cells that protect plants from insects and pathogens. In Arabidopsis, epidermal hairs decrease as internodes increase in height, with only few epidermal hairs produced on the sepals abaxial surface of the early flowers. TRIPTYCHON (TRY) is known to be a negative regulator of epidermal hair development in Arabidopsis, suppressing the formation of ectopic epidermal hairs in the inflorescence. Here, we reported that the second intron of TRY gene plays a critical role in trichome spatial distribution in Arabidopsis. The expression of TRY rises with the increasing stem nodes and reaches the peak in the inflorescence, while the trichomes distribution decrease. The transgenic plants showed that TRY promoter could only drive the genomic instead of coding sequences combined with GUS reporter gene, which indicates that the regulatory elements of TRY expression in inflorescence could be located in the intron regions. Multiple SPLs and MADS-box binding sites were found in the TRY intron2 sequence. Further genetic and biochemistry assays revealed that the flowering-related genes such as SPL9 could bind to these cis-elements directly, contributing to the TRY spatial expression. Since cotton fiber and Arabidopsis trichomes share similar regulatory mechanism, extended analysis showed that the intron2 of cotton TRY genes also contain the cis-elements. Thus, the introns harboring the transcription element may be the general way to regulate the gene expression in different plants and provides molecular clues for the related crops' traits design.

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.

Transcriptional Regulation of miR528 by OsSPL9 Orchestrates Antiviral Response in Rice.

Many microRNAs (miRNAs) are critical regulators of plant antiviral defense. However, little is known about how these miRNAs respond to virus invasion at the transcriptional level. We previously show that defense against Rice stripe virus (RSV) invasion entailed a reduction of miR528 accumulation in rice, alleviating miR528-mediated degradation of L-Ascorbate Oxidase (AO) mRNA and bolstering the antiviral activity of AO. Here we show that the miR528-AO defense module is regulated by the transcription factor SPL9. SPL9 displayed high-affinity binding to specific motifs within the promoter region of miR528 and activated the expression of miR528 gene in vivo. Loss-of-function mutations in SPL9 caused a significant reduction in miR528 accumulation but a substantial increase of AO mRNA, resulting in enhanced plant resistance to RSV. Conversely, transgenic overexpression of SPL9 stimulated the expression of miR528 gene, hence lowering the level of AO mRNA and compromising rice defense against RSV. Importantly, gain in RSV susceptibility did not occur when SPL9 was overexpressed in mir528 loss-of-function mutants, or in transgenic rice expressing a miR528-resistant AO. Taken together, the finding of SPL9-mediated transcriptional activation of miR528 expression adds a new regulatory layer to the miR528-AO antiviral defense pathway.

Transcriptional control of flavonoid biosynthesis: fine-tuning of the MYB-bHLH-WD40 (MBW) complex.

Flavonoids are plant secondary polyphenolic metabolites and fulfil many vital biological functions, offering a valuable metabolic and genetic model for studying transcriptional control of gene expression. Arabidopsis thaliana mainly accumulates 3 types of flavonoids, including flavonols, anthocyanins, and proanthocyanidins (PAs). Flavonoid biosynthesis involves a multitude of well-characterized enzymatic and regulatory proteins. Three R2R3-MYB proteins (MYB11, MYB12, and MYB111) control flavonol biosynthesis via activating the early biosynthetic steps, whereas the production of anthocyanins and PAs requires the MYB-bHLH-WD40 (MBW) complex to activate the late biosynthetic genes. Additional regulators of flavonoid biosynthesis have recently come to light, which interact with R2R3-MYBs or bHLHs to organize or disrupt the formation of the MBW complex, leading to enhanced or compromised flavonoid production. This mini-review gives an overview of how these novel players modulate flavonoid metabolism and thus plant developmental processes and further proposes a fine-tuning mechanism to complete the complex regulatory network controlling flavonoid biosynthesis.