The genetic basis of natural variation in the timing of vegetative phase change in Arabidopsis thaliana.

The juvenile-to-adult transition in plants is known as vegetative phase change and is marked by changes in the expression of leaf traits in response to a decrease in the level of miR156 and miR157. To determine whether this is the only mechanism of vegetative phase change, we measured the appearance of phase-specific leaf traits in 70 natural accessions of Arabidopsis thaliana. We found that leaf shape was poorly correlated with abaxial trichome production (two adult traits), that variation in these traits was not necessarily correlated with the level of miR156, and that there was little to no correlation between the appearance of adult-specific vegetative traits and flowering time. We identified eight quantitative trait loci controlling phase-specific vegetative traits from a cross between the Columbia (Col-0) and Shakdara (Sha) accessions. Only one of these quantitative trait loci includes genes known to regulate vegetative phase change (MIR156A and TOE1), which were expressed at levels consistent with the precocious phenotype of Sha. Our results suggest that vegetative phase change is regulated both by the miR156/SPL module and by genes specific to different vegetative traits, and that natural variation in vegetative phase change can arise from either source.

The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis.

The transition from the juvenile to the adult phase of shoot development in plants is accompanied by changes in vegetative morphology and an increase in reproductive potential. Here, we describe the regulatory mechanism of this transition. We show that miR156 is necessary and sufficient for the expression of the juvenile phase, and regulates the timing of the juvenile-to-adult transition by coordinating the expression of several pathways that control different aspects of this process. miR156 acts by repressing the expression of functionally distinct SPL transcription factors. miR172 acts downstream of miR156 to promote adult epidermal identity. miR156 regulates the expression of miR172 via SPL9 which, redundantly with SPL10, directly promotes the transcription of miR172b. Thus, like the larval-to-adult transition in Caenorhabditis elegans, the juvenile-to-adult transition in Arabidopsis is mediated by sequentially operating miRNAs. miR156 and miR172 are positively regulated by the transcription factors they target, suggesting that negative feedback loops contribute to the stability of the juvenile and adult phases.

VAL genes regulate vegetative phase change via miR156-dependent and independent mechanisms.

How organisms control when to transition between different stages of development is a key question in biology. In plants, epigenetic silencing by Polycomb repressive complex 1 (PRC1) and PRC2 plays a crucial role in promoting developmental transitions, including from juvenile-to-adult phases of vegetative growth. PRC1/2 are known to repress the master regulator of vegetative phase change, miR156, leading to the transition to adult growth, but how this process is regulated temporally is unknown. Here we investigate whether transcription factors in the VIVIPAROUS/ABI3-LIKE (VAL) gene family provide the temporal signal for the epigenetic repression of miR156. Exploiting a novel val1 allele, we found that VAL1 and VAL2 redundantly regulate vegetative phase change by controlling the overall level, rather than temporal dynamics, of miR156 expression. Furthermore, we discovered that VAL1 and VAL2 also act independently of miR156 to control this important developmental transition. In combination, our results highlight the complexity of temporal regulation in plants.

ALTERED MERISTEM PROGRAM1 regulates leaf identity independently of miR156-mediated translational repression.

In Arabidopsis, loss of the carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) produces an increase in the rate of leaf initiation, an enlarged shoot apical meristem and an increase in the number of juvenile leaves. This phenotype is also observed in plants with reduced levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, suggesting that AMP1 might promote SPL activity. However, we found that the amp1 mutant phenotype is only partially corrected by elevated SPL gene expression, and that amp1 has no significant effect on SPL transcript levels, or on the level or the activity of miR156. Although AMP1 has been reported to promote miRNA-mediated translational repression, amp1 did not prevent the translational repression of the miR156 target SPL9 or the miR159 target MYB33. These results suggest that AMP1 regulates vegetative phase change downstream of, or in parallel to, the miR156/SPL pathway, and that it is not universally required for miRNA-mediated translational repression.

Vegetative phase change causes age-dependent changes in phenotypic plasticity.

Phenotypic plasticity allows organisms to optimize traits for their environment. As organisms age, they experience diverse environments that benefit from varying degrees of phenotypic plasticity. Developmental transitions can control these age-dependent changes in plasticity, and as such, the timing of these transitions can determine when plasticity changes in an organism. Here, we investigate how the transition from juvenile-to adult-vegetative development known as vegetative phase change (VPC) contributes to age-dependent changes in phenotypic plasticity and how the timing of this transition responds to environment using both natural accessions and mutant lines in the model plant Arabidopsis thaliana. We found that the adult phase of vegetative development has greater plasticity in leaf morphology than the juvenile phase and confirmed that this difference in plasticity is caused by VPC using mutant lines. Furthermore, we found that the timing of VPC, and therefore the time when increased plasticity is acquired, varies significantly across genotypes and environments. The consistent age-dependent changes in plasticity caused by VPC suggest that VPC may be adaptive. This genetic and environmental variation in the timing of VPC indicates the potential for population-level adaptive evolution of VPC.

Low light intensity delays vegetative phase change.

Plants that develop under low light (LL) intensity often display a phenotype known as the "shade tolerance syndrome (STS)". This syndrome is similar to the phenotype of plants in the juvenile phase of shoot development, but the basis for this similarity is unknown. We tested the hypothesis that the STS is regulated by the same mechanism that regulates the juvenile vegetative phase by examining the effect of LL on rosette development in Arabidopsis (Arabidopsis thaliana). We found that LL prolonged the juvenile vegetative phase and that this was associated with an increase in the expression of the master regulators of vegetative phase change, miR156 and miR157, and a decrease in the expression of their SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) targets. Exogenous sucrose partially corrected the effect of LL on seedling development and miR156 expression. Our results suggest that the response of Arabidopsis to LL is mediated by an increase in miR156/miR157 expression and by factors that repress SPL gene expression independently of miR156/miR157, and is caused in part by a decrease in carbohydrate production. The effect of LL on vegetative phase change does not require the photoreceptors and transcription factors responsible for the shade avoidance syndrome, implying that light intensity and light quality regulate rosette development through different pathways.

The carbon economics of vegetative phase change.

Across plant species and biomes, a conserved set of leaf traits govern the economic strategy used to assimilate and invest carbon. As plants age, they face new challenges that may require shifts in this leaf economic strategy. In this study, we investigate the role of the developmental transition, vegetative phase change (VPC), in altering carbon economics as plants age. We used overexpression of microRNA 156 (miR156), the master regulator of VPC, to modulate the timing of VPC in Populus tremula x alba, Arabidopsis thaliana and Zea mays to understand the impact of this transition on leaf economic traits, including construction cost, payback time and return on investment. Here, we find that VPC causes a shift from a low-cost, quick return juvenile strategy to a high-cost, high-return adult strategy. The juvenile strategy is advantageous in light-limited conditions, whereas the adult strategy provides greater returns in high light. The transition between these strategies is correlated with the developmental decline in the level of miR156, suggesting that is regulated by the miR156/SPL pathway. Our results provide an ecophysiological explanation for the existence of juvenile and adult leaf types and suggest that natural selection for these alternative economic strategies could be an important factor in plant evolution.

Role for the shoot apical meristem in the specification of juvenile leaf identity in Arabidopsis.

The extent to which the shoot apical meristem (SAM) controls developmental decisions, rather than interpreting them, is a longstanding issue in plant development. Previous work suggests that vegetative phase change is regulated by signals intrinsic and extrinsic to the SAM, but the relative importance of these signals for this process is unknown. We investigated this question by examining the effect of meristem-deficient mutations on vegetative phase change and on the expression of key regulators of this process, miR156 and its targets, SPL transcription factors. We found that the precocious phenotypes of meristem-deficient mutants are a consequence of reduced miR156 accumulation. Tissue-specific manipulation of miR156 levels revealed that the SAM functions as an essential pool of miR156 early in shoot development, but that its effect on leaf identity declines with age. We also found that SPL genes control meristem size by repressing WUSCHEL expression via a novel genetic pathway.

Development and evolution of age-dependent defenses in ant-acacias.

Age-dependent changes in plant defense against herbivores are widespread, but why these changes exist remains a mystery. We explored this question by examining a suite of traits required for the interaction between swollen thorn acacias (genus Vachellia) and ants of the genus Pseudomyrmex In this system, plants provide ants with refuge and food in the form of swollen stipular spines, protein-lipid-rich "Beltian" bodies, and sugar-secreting extrafloral nectaries-the "swollen thorn syndrome." We show that this syndrome develops at a predictable time in shoot development and is tightly associated with the temporal decline in the microRNAs miR156 and miR157 and a corresponding increase in their targets-the SPL transcription factors. Growth under reduced light intensity delays both the decline in miR156/157 and the development of the swollen thorn syndrome, supporting the conclusion that these traits are controlled by the miR156-SPL pathway. Production of extrafloral nectaries by Vachellia sp. that do not house ants is also correlated with a decline in miR156/157, suggesting that this syndrome evolved by co-opting a preexisting age-dependent program. Along with genetic evidence from other model systems, these findings support the hypothesis that the age-dependent development of the swollen thorn syndrome is a consequence of genetic regulation rather than a passive developmental pattern arising from developmental constraints on when these traits can develop.

H2A.Z promotes the transcription of MIR156A and MIR156C in Arabidopsis by facilitating the deposition of H3K4me3.

Vegetative phase change in Arabidopsis thaliana is mediated by a decrease in the level of MIR156A and MIR156C, resulting in an increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Changes in chromatin structure are required for the downregulation of MIR156A and MIR156C, but whether chromatin structure contributes to their initial elevated expression is unknown. We found that mutations in components of the SWR1 complex (ARP6, SEF) and in genes encoding H2A.Z (HTA9 and HTA11) reduce the expression of MIR156A and MIR156C, and accelerate vegetative phase change, indicating that H2A.Z promotes juvenile vegetative identity. However, arp6 and sef did not accelerate the temporal decline in miR156, and the downregulation of MIR156A and MIR156C was not accompanied by significant change in the level of H2A.Z at these loci. We conclude that H2A.Z contributes to the high expression of MIR156A/MIR156C early in shoot development, but does not regulate the timing of vegetative phase change. Our results also suggest that H2A.Z promotes the expression of MIR156A/MIR156C by facilitating the deposition of H3K4me3, rather than by decreasing nucleosome occupancy.

MicroRNA156-mediated changes in leaf composition lead to altered photosynthetic traits during vegetative phase change.

Plant morphology and physiology change with growth and development. Some of these changes are due to change in plant size and some are the result of genetically programmed developmental transitions. In this study we investigate the role of the developmental transition, vegetative phase change (VPC), on morphological and photosynthetic changes. We used overexpression of microRNA156, the master regulator of VPC, to modulate the timing of VPC in Populus tremula × alba, Zea mays, and Arabidopsis thaliana to determine its role in trait variation independent of changes in size and overall age. Here, we find that juvenile and adult leaves in all three species photosynthesize at different rates and that these differences are due to phase-dependent changes in specific leaf area (SLA) and leaf N but not photosynthetic biochemistry. Further, we found juvenile leaves with high SLA were associated with better photosynthetic performance at low light levels. This study establishes a role for VPC in leaf composition and photosynthetic performance across diverse species and environments. Variation in leaf traits due to VPC are likely to provide distinct benefits under specific environments; as a result, selection on the timing of this transition could be a mechanism for environmental adaptation.

Vegetative phase change in Populus tremula × alba.

Plants transition through juvenile and adult phases of vegetative development in a process known as vegetative phase change (VPC). In poplars (genus Populus) the differences between these stages are subtle, making it difficult to determine when this transition occurs. Previous studies of VPC in poplars have relied on plants propagated in vitro, leaving the natural progression of this process unknown. We examined developmental morphology of seed-grown and in vitro derived Populus tremula × alba (clone 717-1B4), and compared the phenotype of these to transgenics with manipulated miR156 expression, the master regulator of VPC. In seed-grown plants, most traits changed from node-to-node during the first 3 months of development but remained constant after node 25. Many traits remained unchanged in clones over-expressing miR156, or were enhanced when miR156 was lowered, demonstrating their natural progression is regulated by the miR156/SPL pathway. The characteristic leaf fluttering of Populus is one of these miR156-regulated traits. Vegetative development in plants grown from culture mirrored that of seed-grown plants, allowing direct comparison between plants often used in research and those found in nature. These results provide a foundation for further research on the role of VPC in the ecology and evolution of this economically important genus.

Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana.

Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression.

Repression of miR156 by miR159 Regulates the Timing of the Juvenile-to-Adult Transition in Arabidopsis.

Temporally regulated microRNAs have been identified as master regulators of developmental timing in both animals and plants. In plants, vegetative development is regulated by a temporal decrease in miR156 level, but how this decreased expression is initiated and then maintained during shoot development remains elusive. Here, we show that miR159 is required for the correct timing of vegetative development in Arabidopsis thaliana Loss of miR159 increases miR156 level throughout shoot development and delays vegetative development, whereas overexpression of miR159 slightly accelerated vegetative development. The repression of miR156 by miR159 is predominantly mediated by MYB33, an R2R3 MYB domain transcription factor targeted by miR159. Loss of MYB33 led to subtle precocious vegetative phase change phenotypes in spite of the significant downregulation of miR156. MYB33 simultaneously promotes the transcription of MIR156A and MIR156C, as well as their target, SPL9, by directly binding to the promoters of these three genes. Rather than acting as major players in vegetative phase change in Arabidopsis, our results suggest that miR159 and MYB33 function as modifiers of vegetative phase change; i.e., miR159 facilitates vegetative phase change by repressing MYB33 expression, thus preventing MYB33 from hyperactivating miR156 expression throughout shoot development to ensure correct timing of the juvenile-to-adult transition in Arabidopsis.

Ian Sussex: simple tools, clever experiments and new insights into plant development.

Ian Sussex, who began his career at a time when the most powerful tool available to plant developmental biologists was a scalpel, helped transform the discipline of plant developmental biology into the dynamic, sophisticated field that it is today. He did this through his own research, through an influential book that he wrote with his friend and colleague Taylor Steeves, and through his many students and post-doctoral fellows, to whom he gave the greatest gift a scientist can receive - the freedom to do whatever they wanted.

Epigenetic Regulation of Vegetative Phase Change in Arabidopsis.

Vegetative phase change in flowering plants is regulated by a decrease in the level of miR156. The molecular mechanism of this temporally regulated decrease in miR156 expression is still unknown. Most of the miR156 in Arabidopsis thaliana shoots is produced by MIR156A and MIR156C. We found that the downregulation of these genes during vegetative phase change is associated with an increase in their level of histone H3 lysine 27 trimethylation (H3K27me3) and requires this chromatin modification. The increase in H3K27me3 at MIR156A/MIR156C is associated with an increase in the binding of PRC2 to these genes and is mediated redundantly by the E(z) homologs SWINGER and CURLY LEAF. The CHD3 chromatin remodeler PICKLE (PKL) promotes the addition of H3K27me3 to MIR156A/MIR156C but is not responsible for the temporal increase in this chromatin mark. PKL is bound to the promoters of MIR156A/MIR156C, where it promotes low levels of H3K27ac early in shoot development and stabilizes the nucleosome at the +1 position. These results suggest a molecular mechanism for the initiation and maintenance of vegetative phase change in plants.

Developmental Functions of miR156-Regulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) Genes in Arabidopsis thaliana.

Correct developmental timing is essential for plant fitness and reproductive success. Two important transitions in shoot development-the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition-are mediated by a group of genes targeted by miR156, SQUAMOSA PROMOTER BINDING PROTEIN (SBP) genes. To determine the developmental functions of these genes in Arabidopsis thaliana, we characterized their expression patterns, and their gain-of-function and loss-of-function phenotypes. Our results reveal that SBP-LIKE (SPL) genes in Arabidopsis can be divided into three functionally distinct groups: 1) SPL2, SPL9, SPL10, SPL11, SPL13 and SPL15 contribute to both the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition, with SPL9, SP13 and SPL15 being more important for these processes than SPL2, SPL10 and SPL11; 2) SPL3, SPL4 and SPL5 do not play a major role in vegetative phase change or floral induction, but promote the floral meristem identity transition; 3) SPL6 does not have a major function in shoot morphogenesis, but may be important for certain physiological processes. We also found that miR156-regulated SPL genes repress adventitious root development, providing an explanation for the observation that the capacity for adventitious root production declines as the shoot ages. miR156 is expressed at very high levels in young seedlings, and declines in abundance as the shoot develops. It completely blocks the expression of its SPL targets in the first two leaves of the rosette, and represses these genes to different degrees at later stages of development, primarily by promoting their translational repression. These results provide a framework for future studies of this multifunctional family of transcription factors, and offer new insights into the role of miR156 in Arabidopsis development.

The Arabidopsis Mediator CDK8 module genes CCT (MED12) and GCT (MED13) are global regulators of developmental phase transitions.

Temporal coordination of developmental programs is necessary for normal ontogeny, but the mechanism by which this is accomplished is still poorly understood. We have previously shown that two components of the Mediator CDK8 module encoded by CENTER CITY (CCT; Arabidopsis MED12) and GRAND CENTRAL (GCT; Arabidopsis MED13) are required for timing of pattern formation during embryogenesis. A morphological, molecular and genomic analysis of the post-embryonic phenotype of gct and cct mutants demonstrated that these genes also promote at least three subsequent developmental transitions: germination, vegetative phase change, and flowering. Genetic and molecular analyses indicate that GCT and CCT operate in parallel to gibberellic acid, a phytohormone known to regulate these same three transitions. We demonstrate that the delay in vegetative phase change in gct and cct is largely due to overexpression of miR156, and that the delay in flowering is due in part to upregulation of FLC. Thus, GCT and CCT coordinate vegetative and floral transitions by repressing the repressors miR156 and FLC. Our results suggest that MED12 and MED13 act as global regulators of developmental timing by fine-tuning the expression of temporal regulatory genes.

3' fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3.

trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)-mediated cleavage of tasiRNA gene (TAS) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1-RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3' cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3' cleavage fragment. When the 3' nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3' cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that (i) SGS3 associates with AGO1-RISC via the double-stranded RNA formed by the 3'-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1-RISC molecular surface, (ii) SGS3 protects the 3' cleavage fragment of TAS2 RNA from degradation, and (iii) the observed SGS3-dependent stabilization of the 3' fragment of TAS2 RNA is key to tasiRNA production.