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An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)-MKK4/MKK5-MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4DD or MKK5DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development. MPK3/MPK6 cascade functions downstream of ERECTA family receptors in the epidermis and upstream of the STOMAGEN peptide in the mesophyll tissue to control stomatal development. [ABSTRACT FROM AUTHOR]

The biological function of RNA can be modulated by base modifications. Here, we unveiled the occurrence of N4-acetylation of cytidine in plant RNA, including mRNA, by employing LC-MS/MS and acRIP-seq. We identified 325 acetylated transcripts from the leaves of 4-week-old Arabidopsis (Arabidopsis thaliana) plants and determined that 2 partially redundant N-ACETYLTRANSFERASEs FOR CYTIDINE IN RNA (ACYR1 and ACYR2), which are homologous to mammalian NAT10, are required for acetylating RNA in vivo. A double-null mutant was embryo lethal, while eliminating 3 of the 4 ACYR alleles led to defects in leaf development. These phenotypes could be traced back to the reduced acetylation and concomitant destabilization of the transcript of TOUGH, which is required for miRNA processing. These findings indicate that N4-acetylation of cytidine is a modulator of RNA function with a critical role in plant development and likely many other processes. The base modification N4-acetylation of cytidine in RNA is essential for microRNA biogenesis as well as leaf and embryo development in plants. [ABSTRACT FROM AUTHOR]

C3 and C4 grasses directly and indirectly provide the vast majority of calories to the human diet, yet our understanding of the molecular mechanisms driving photosynthetic productivity in grasses is largely unexplored. Ground meristem cells divide to form mesophyll or vascular initial cells early in leaf development in C3 and C4 grasses. Here we define a genetic circuit composed of SHORT ROOT (SHR), INDETERMINATE DOMAIN (IDD), and PIN-FORMED (PIN) family members that specifies vascular identify and ground cell proliferation in leaves of both C3 and C4 grasses. Ectopic expression and loss-of-function mutant studies of SHR paralogs in the C3 plant Oryza sativa (rice) and the C4 plant Setaria viridis (green millet) revealed the roles of these genes in both minor vein formation and ground cell differentiation. Genetic and in vitro studies further suggested that SHR regulates this process through its interactions with IDD12 and 13. We also revealed direct interactions of these IDD proteins with a putative regulatory element within the auxin transporter gene PIN5c. Collectively, these findings indicate that a SHR–IDD regulatory circuit mediates auxin transport by negatively regulating PIN expression to modulate minor vein patterning in the grasses. A SHORT ROOT–INTERMEDIATE DOMAIN regulatory circuit modulates auxin transport by negatively regulating PIN-FORMED expression to regulate minor vein patterning in grasses. [ABSTRACT FROM AUTHOR]

Signals emanating from chloroplasts influence nuclear gene expression, but roles of retrograde signals during chloroplast development are unclear. To address this gap, we analyzed transcriptomes of non-photosynthetic maize mutants and compared them to transcriptomes of stages of normal leaf development. The transcriptomes of two albino mutants lacking plastid ribosomes resembled transcriptomes at very early stages of normal leaf development, whereas the transcriptomes of two chlorotic mutants with thylakoid targeting or plastid transcription defects resembled those at a slightly later stage. We identified ∼2,700 differentially expressed genes, which fall into six major categories based on the polarity and mutant-specificity of the change. Downregulated genes were generally expressed late in normal development and were enriched in photosynthesis genes, whereas upregulated genes act early and were enriched for functions in chloroplast biogenesis and cytosolic translation. We showed further that target-of-rapamycin (TOR) signaling was elevated in mutants lacking plastid ribosomes and declined in concert with plastid ribosome buildup during normal leaf development. Our results implicate three plastid signals as coordinators of photosynthetic differentiation. One signal requires plastid ribosomes and activates photosynthesis genes. A second signal reflects attainment of chloroplast maturity and represses chloroplast biogenesis genes. A third signal, the consumption of nutrients by developing chloroplasts, represses TOR, promoting termination of cell proliferation during leaf development. [ABSTRACT FROM AUTHOR]

The plant-specific family of WUSCHEL (WUS)-related homeobox (WOX) transcription factors is key regulators of embryogenesis, meristem maintenance, and lateral organ development in flowering plants. The modern/WUS clade transcriptional repressor STENOFOLIA/LAMINA1(LAM1), and the intermediate/WOX9 clade transcriptional activator MtWOX9/NsWOX9 antagonistically regulate leaf blade expansion, but the molecular mechanism is unknown. Using transcriptome profiling and biochemical methods, we determined that NsCKX3 is the common target of LAM1 and NsWOX9 in Nicotiana sylvestris. LAM1 and NsWOX9 directly recognize and bind to the same cis-elements in the NsCKX3 promoter to repress and activate its expression, respectively, thus controlling the levels of active cytokinins in vivo. Disruption of NsCKX3 in the lam1 background yielded a phenotype similar to the knockdown of NsWOX9 in lam1, while overexpressing NsCKX3 resulted in narrower and shorter lam1 leaf blades reminiscent of NsWOX9 overexpression in the lam1 mutant. Moreover, we established that LAM1 physically interacts with NsWOX9, and this interaction is required to regulate NsCKX3 transcription. Taken together, our results indicate that repressor and activator WOX members oppositely regulate a common downstream target to function in leaf blade outgrowth, offering a novel insight into the role of local cytokinins in balancing cell proliferation and differentiation during lateral organ development. [ABSTRACT FROM AUTHOR]

The basic mechanisms of leaf development have been revealed through a combination of genetics and intense analyses in select model species. The genetic basis for diversity in leaf morphology seen in nature is also being unraveled through recent advances in techniques and technologies related to genomics and transcriptomics, which have had a major impact on these comparative studies. However, this has led to the emergence of new unresolved questions about the mechanisms that generate the diversity of leaf form. Here, we provide a review of the current knowledge of the fundamental molecular genetic mechanisms underlying leaf development with an emphasis on natural variation and conserved gene regulatory networks involved in leaf development. Beyond that, we discuss open questions/enigmas in the area of leaf development, how recent technologies can best be deployed to generate a unified understanding of leaf diversity and its evolution, and what untapped fields lie ahead. [ABSTRACT FROM AUTHOR]

During leaf senescence, the final stage of leaf development, nutrients are recycled from leaves to other organs, and therefore proper control of senescence is thus critical for plant fitness. Although substantial progress has been achieved in understanding leaf senescence in annual plants, the molecular factors that control leaf senescence in perennial woody plants are largely unknown. Using RNA sequencing, we obtained a high-resolution temporal profile of gene expression during autumn leaf senescence in poplar (Populus tomentosa). Identification of hub transcription factors (TFs) by co-expression network analysis of genes revealed that senescence-associated NAC family TFs (Sen-NAC TFs) regulate autumn leaf senescence. Age-dependent alternative splicing (AS) caused an intron retention (IR) event in the pre-mRNA encoding PtRD26, a NAC-TF. This produced a truncated protein PtRD26IR, which functions as a dominant-negative regulator of senescence by interacting with multiple hub Sen-NAC TFs, thereby repressing their DNA-binding activities. Functional analysis of senescence-associated splicing factors identified two U2 auxiliary factors that are involved in AS of PtRD26IR. Correspondingly, silencing of these factors decreased PtRD26IR transcript abundance and induced early senescence. We propose that an age-dependent increase of IR splice variants derived from Sen-NAC TFs is a regulatory program to fine tune the molecular mechanisms that regulate leaf senescence in trees. [ABSTRACT FROM AUTHOR]

After phragmoplast expansion is complete, the cell plate fuses with the parental plasma membrane and existing cell wall. Phragmoplast expansion occurs in distinct stages encompassing the integrity of phragmoplast and early compared with late phragmoplast guidance, but the precise mechanisms and protein-protein interactions that promote correct phragmoplast guidance through theses stages remain elusive. [Extracted from the article]

Alkenes are linear hydrocarbons with one or more double bonds. Despite their potential as biofuels and precursors for specialty chemicals, the underlying biochemistry and genetics of alkene biosynthesis in plants remain elusive. Here, we report on a screen of natural accessions of poplar (Populus trichocarpa), revealing that the leaf cuticular waxes are predominantly composed of alkanes and alkenes. Interestingly, the accumulation of alkenes increases with leaf development, is limited to the abaxial side of the leaf, and is impaired in a few accessions. Among other genes, a β-ketoacyl CoA synthase gene (PotriKCS1) was downregulated in leaves from non-alkene-producing accessions. We demonstrated biochemically that PotriKCS1 elongates monounsaturated fatty acids and is responsible for the recruitment of unsaturated substrates to the cuticular wax. Moreover, we found significant associations between the presence of alkenes and tree growth and resistance to leaf spot. These findings highlight the crucial role of cuticular waxes as the first point of contact with the environment, and they provide a foundation for engineering long-chain monounsaturated oils in other species. [ABSTRACT FROM AUTHOR]

The developmental plasticity of leaf size and shape is important for leaf function and plant survival. However, the mechanisms by which plants form diverse leaves in response to environmental conditions are not well understood. Here, we identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1) and found that it regulates leaf development by promoting the degradation of TCP INTERACTOR-CONTAINING EAR MOTIF PROTEIN1 (TIE1), an important repressor of CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, which are key for leaf development. TEAR1 contains a typical C3H2C3-type RING domain and has E3 ligase activity. We show that TEAR1 interacts with the TCP repressor TIE1, which is ubiquitinated in vivo and degraded by the 26S proteasome system. We demonstrate that TEAR1 is colocalized with TIE1 in nuclei and negatively regulates TIE1 protein levels. Overexpression of TEAR1 rescued leaf defects caused by TIE1 overexpression, whereas disruption of TEAR1 resulted in leaf phenotypes resembling those caused by TIE1 overexpression or TCP dysfunction. Deficiency in TEAR partially rescued the leaf defects of TCP4 overexpression line and enhanced the wavy leaf phenotypes of jaw-5D. We propose that TEAR1 positively regulates CIN-like TCP activity to promote leaf development by mediating the degradation of the TCP repressor TIE1. [ABSTRACT FROM AUTHOR]

We applied 15N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome. [ABSTRACT FROM AUTHOR]

Leaf morphogenesis and differentiation are highly flexible processes, resulting in a large diversity of leaf forms. The development of compound leaves involves an extended morphogenesis stage compared with that of simple leaves, and the tomato (Solanum lycopersicum) mutant clausa (clau) exposes a potential for extended morphogenesis in tomato leaves. Here, we report that the CLAU gene encodes a MYB transcription factor that has evolved a unique role in compound-leaf species to promote an exit from the morphogenetic phase of tomato leaf development. We show that CLAU attenuates cytokinin signaling, and that clau plants have increased cytokinin sensitivity. The results suggest that flexible leaf patterning involves a coordinated interplay between transcription factors and hormones. [ABSTRACT FROM AUTHOR]

Flattened leaf architecture is not a default state but depends on positional information to precisely coordinate patterns of cell division in the growing primordium. This information is provided, in part, by the boundary between the adaxial (top) and abaxial (bottom) domains of the leaf, which are specified via an intricate gene regulatory network whose precise circuitry remains poorly defined. Here, we examined the contribution of the ASYMMETRIC LEAVES (AS) pathway to adaxial-abaxial patterning in Arabidopsis thaliana and demonstrate that AS1-AS2 affects this process via multiple, distinct regulatory mechanisms. AS1-AS2 uses Polycomb-dependent and -independent mechanisms to directly repress the abaxial determinants MIR166A , YABBY5 , and AUXIN RESPONSE FACTOR3 (ARF3), as well as a nonrepressive mechanism in the regulation of the adaxial determinant TAS3A. These regulatory interactions, together with data from prior studies, lead to a model in which the sequential polarization of determinants, including AS1-AS2, explains the establishment and maintenance of adaxial-abaxial leaf polarity. Moreover, our analyses show that the shared repression of ARF3 by the AS and trans -acting small interfering RNA (ta-siRNA) pathways intersects with additional AS1-AS2 targets to affect multiple nodes in leaf development, impacting polarity as well as leaf complexity. These data illustrate the surprisingly multifaceted contribution of AS1-AS2 to leaf development showing that, in conjunction with the ta-siRNA pathway, AS1-AS2 keeps the Arabidopsis leaf both flat and simple. [ABSTRACT FROM AUTHOR]

The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens , can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM , SMR1/LGO , and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2 , KANADI1 and 3 , and YABBY2 , 3 , and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1 , APETALA3 , and the MADS box gene AGL24 , whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3 , SEPALLATA3 , LEAFY , UNUSUAL FLORAL ORGANS , TERMINAL FLOWER1 , AGAMOUS-LIKE24 , and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana , the α/β-fold hydrolase KARRIKIN INSENSITIVE2 (KAI2) is essential for normal seed germination, seedling development, and leaf morphogenesis, as well as for responses to karrikins. KAI2 is a paralog of DWARF14 (D14), the proposed strigolactone receptor, but the evolutionary timing of functional divergence between the KAI2 and D14 clades has not been established. By swapping gene promoters, we show that Arabidopsis KAI2 and D14 proteins are functionally distinct. We show that the catalytic serine of KAI2 is essential for function in plants and for biochemical activity in vitro. We identified two KAI2 homologs from Selaginella moellendorffii and two from Marchantia polymorpha. One from each species could hydrolyze the strigolactone analog GR24 in vitro, but when tested for their ability to complement Arabidopsis d14 and kai2 mutants, neither of these homologs was effective. However, the second KAI2 homolog from S. moellendorffii was able to complement the seedling and leaf development phenotypes of Arabidopsis kai2. This homolog could not transduce signals from exogenous karrikins, strigolactone analogs, or carlactone, but its activity did depend on the conserved catalytic serine. We conclude that KAI2, and most likely the endogenous signal to which it responds, has been conserved since the divergence of lycophytes and angiosperm lineages, despite their major developmental and morphogenic differences. [ABSTRACT FROM AUTHOR]

Plants initiate leaf senescence to relocate nutrients and energy from aging leaves to developing tissues or storage organs for growth, reproduction, and defense. Leaf senescence, the final stage of leaf development, is regulated by various environmental stresses, developmental cues, and endogenous hormone signals. Jasmonate (JA), a lipid-derived phytohormone essential for plant defense and plant development, serves as an important endogenous signal to activate senescence-associated gene expression and induce leaf senescence. This study revealed one of the mechanisms underlying JA -induced leaf senescence: antagonistic interactions of the bHLH subgroup IIIe factors MYC2, MYC3, and MYC4 with the bHLH subgroup IIId factors bHLH03, bHLH13, bHLH14, and bHLH17. We showed that MYC2, MYC3, and MYC4 function redundantly to activate JA -induced leaf senescence. MYC2 binds to and activates the promoter of its target gene SAG29 (SENESCENCE-ASSOCIATED GENE29) to activate JA -induced leaf senescence. Interestingly, plants have evolved an elaborate feedback regulation mechanism to modulate JA -induced leaf senescence: The bHLH subgroup IIId factors (bHLH03, bHLH13, bHLH14, and bHLH17) bind to the promoter of SAG29 and repress its expression to attenuate MYC2/MYC3/MYC4-activated JA -induced leaf senescence. The antagonistic regulation by activators and repressors would mediate JA -induced leaf senescence at proper level suitable for plant survival in fluctuating environmental conditions. [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts. [ABSTRACT FROM AUTHOR]

In addition to acting as photoprotective compounds, carotenoids also serve as precursors in the biosynthesis of several phytohormones and proposed regulatory signals. Here, we report a signaling process derived from carotenoids that regulates early chloroplast and leaf development. Biosynthesis of the signal depends on ζ-carotene desaturase activity encoded by the ζ-CAROTENE DESATURASE (ZDS)/ CHLOROPLAST BIOGENESIS5 (CLB5) gene in Arabidopsis thaliana. Unlike other carotenoid-deficient plants, zds / clb5 mutant alleles display profound alterations in leaf morphology and cellular differentiation as well as altered expression of many plastid- and nucleus-encoded genes. The leaf developmental phenotypes and gene expression alterations of zds / clb5 / spc1 / pde181 plants are rescued by inhibitors or mutations of phytoene desaturase, demonstrating that phytofluene and/or ζ-carotene are substrates for an unidentified signaling molecule. Our work further demonstrates that this signal is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4. [ABSTRACT FROM AUTHOR]

Class I KNOTTED -like homeobox (KNOXI) genes are critical for the maintenance of the shoot apical meristem. The expression domain of KNOXI is regulated by ASYMMETRIC LEAVES1 / ROUGHSHEATH2 / PHANTASTICA (ARP) genes, which are associated with leaf morphology. In the inverted repeat-lacking clade (IRLC) of Fabaceae, the orthologs of LEAFY (LFY) function in place of KNOXI to regulate compound leaf development. Here, we characterized loss-of-function mutants of ARP (PHAN) and SHOOTMERISTEMLESS (STM) - and BREVIPEDICELLUS (BP) -like KNOXI in the model IRLC legume species Medicago truncatula. The function of ARP genes is species specific. The repression of STM / BP-like KNOXI genes in leaves is not mediated by PHAN , and no suppression of PHAN by STM/BP-like KNOXI genes was observed either, indicating that STM/BP-like KNOXI genes are uncoupled from PHAN in M. truncatula. Furthermore, comparative analyses of phenotypic output in response to ectopic expression of KNOXI and the M. truncatula LFY ortholog, SINGLE LEAFLET1 (SGL1), reveal that KNOXI and SGL1 regulate parallel pathways in leaf development. We propose that SGL1 probably functions in a stage-specific manner in the regulation of the indeterminate state of developing leaves in M. truncatula. [ABSTRACT FROM AUTHOR]

This article explores the fate of plastid DNA (ptDNA) during leaf development. The authors respond to a critique of their previous findings, arguing that the decline in ptDNA occurs during proplastid-to-chloroplast development, rather than in mature-to-senescent leaf stages. They emphasize the importance of considering both the quantity and quality of ptDNA when assessing its functional relevance. The authors also refute claims made by another study regarding the presence of leaky plastid membranes and the reliability of DAPI-DNA quantification. They conclude that the absence of detectable DAPI-DNA fluorescence in some plastids is due to a decrease in ptDNA content and/or molecular integrity as leaves develop. The document also discusses the size and copy number of plastids in maize seedlings during different stages of development, and addresses the question of whether chloroplasts can conduct photosynthesis without plastid DNA. Detailed methods for preparing and imaging maize tissue, cells, and plastids for fluorescence microscopy are provided. [Extracted from the article]

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed high-resolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana , sugar beet [ Beta vulgaris ], tobacco [ Nicotiana tabacum ], and maize [ Zea mays ]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis , tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ∼30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation. [ABSTRACT FROM AUTHOR]

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5 , GRF6 , CYTOKININ RESPONSE FACTOR2 , CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5 , GRF3 , COL5 , and ARR4 , and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf. [ABSTRACT FROM AUTHOR]

Leaf shape elaboration and organ separation are critical for plant morphogenesis. We characterized the developmental roles of LOBED LEAFLET1 by analyzing a recessive mutant in the model legume Medicago truncatula. An ortholog of Arabidopsis thaliana ARGONAUTE7 (AGO7), Mt -AGO7/LOBED LEAFLET1, is required for the biogenesis of a trans -acting short interfering RNA (ta-siRNA) to negatively regulate the expression of AUXIN RESPONSE FACTORs in M. truncatula. Loss of function in AGO7 results in pleiotropic phenotypes in different organs. The prominent phenotype of the ago7 mutant is lobed leaf margins and more widely spaced lateral organs, suggesting that the trans -acting siRNA3 (TAS3) pathway negatively regulates the formation of boundaries and the separation of lateral organs in M. truncatula. Genetic interaction analysis with the smooth leaf margin1 (slm1) mutant revealed that leaf margin formation is cooperatively regulated by the auxin/SLM1 (ortholog of Arabidopsis PIN-FORMED1) module, which influences the initiation of leaf margin teeth, and the TAS3 ta-siRNA pathway, which determines the degree of margin indentation. Further investigations showed that the TAS3 ta-siRNA pathway and NO APICAL MERISTEM (ortholog of Arabidopsis CUP-SHAPED COTYLEDON) antagonistically regulate both leaf margin development and lateral organ separation, and the regulation is partially dependent on the auxin/SLM1 module. [ABSTRACT FROM AUTHOR]

We performed large-scale, quantitative analyses of the maize (Zea mays) leaf proteome and phosphoproteome at four developmental stages. Exploiting the developmental gradient of maize leaves, we analyzed protein and phosphoprotein abundance as maize leaves transition from proliferative cell division to differentiation to cell expansion and compared these developing zones to one another and the mature leaf blade. Comparison of the proteomes and phosphoproteomes suggests a key role for posttranslational regulation in developmental transitions. Analysis of proteins with cell wall– and hormone-related functions illustrates the utility of the data set and provides further insight into maize leaf development. We compare phosphorylation sites identified here to those previously identified in Arabidopsis thaliana. We also discuss instances where comparison of phosphorylated and unmodified peptides from a particular protein indicates tissue-specific phosphorylation. For example, comparison of unmodified and phosphorylated forms of PINFORMED1 (PIN1) suggests a tissue-specific difference in phosphorylation, which correlates with changes in PIN1 polarization in epidermal cells during development. Together, our data provide insights into regulatory processes underlying maize leaf development and provide a community resource cataloging the abundance and phosphorylation status of thousands of maize proteins at four leaf developmental stages. [ABSTRACT FROM AUTHOR]

The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities. [ABSTRACT FROM AUTHOR]

Flexible maturation rates underlie part of the diversity of leaf shape, and tomato (Solanum lycopersicum) leaves are compound due to prolonged organogenic activity of the leaf margin. The CINCINNATA -TEOSINTE BRANCHED1, CYCLOIDEA, PCF (CIN-TCP) transcription factor LANCEOLATE (LA) restricts this organogenic activity and promotes maturation. Here, we show that tomato APETALA1/FRUITFULL (AP1/FUL) MADS box genes are involved in tomato leaf development and are repressed by LA. AP1/FUL expression is correlated negatively with LA activity and positively with the organogenic activity of the leaf margin. LA binds to the promoters of the AP1/FUL genes MBP20 and TM4. Overexpression of MBP20 suppressed the simple-leaf phenotype resulting from upregulation of LA activity or from downregulation of class I knotted like homeobox (KNOXI) activity. Overexpression of a dominant-negative form of MBP20 led to leaf simplification and partly suppressed the increased leaf complexity of plants with reduced LA activity or increased KNOXI activity. Tomato plants overexpressing miR319, a negative regulator of several CIN-TCP genes including LA , flower with fewer leaves via an SFT-dependent pathway, suggesting that miR319-sensitive CIN-TCPs delay flowering in tomato. These results identify a role for AP1/FUL genes in vegetative development and show that leaf and plant maturation are regulated via partially independent mechanisms. [ABSTRACT FROM AUTHOR]

Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development. [ABSTRACT FROM AUTHOR]

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ. [ABSTRACT FROM AUTHOR]

Position-dependent patterning of hair and non-hair cells in the Arabidopsis thaliana root epidermis is a powerful system to study the molecular basis of cell fate specification. Here, we report an epidermal patterning mutant affecting the ADENOSINE DIMETHYL TRANSFERASE 1A (DIM1A) rRNA dimethylase gene, predicted to participate in rRNA posttranscriptional processing and base modification. Consistent with a role in ribosome biogenesis, DIM1A is preferentially expressed in regions of rapid growth, and its product is nuclear localized with nucleolus enrichment. Furthermore, DIM1A preferentially accumulates in the developing hair cells, and the dim1A point mutant alters the cell-specific expression of the transcriptional regulators GLABRA2 , CAPRICE , and WEREWOLF. Together, these findings suggest that establishment of cell-specific gene expression during root epidermis development is dependent upon proper ribosome biogenesis, possibly due to the sensitivity of the cell fate decision to relatively small differences in gene regulatory activities. Consistent with its effect on the predicted S -adenosyl- l -Met binding site, dim1A plants lack the two 18S rRNA base modifications but exhibit normal pre-rRNA processing. In addition to root epidermal defects, the dim1A mutant exhibits abnormal root meristem division, leaf development, and trichome branching. Together, these findings provide new insights into the importance of rRNA base modifications and translation regulation for plant growth and development. [ABSTRACT FROM AUTHOR]

In a majority of species, leaf development is thought to proceed in a bilaterally symmetric fashion without systematic asymmetries. This is despite the left and right sides of an initiating primordium occupying niches that differ in their distance from sinks and sources of auxin. Here, we revisit an existing model of auxin transport sufficient to recreate spiral phyllotactic patterns and find previously overlooked asymmetries between auxin distribution and the centers of leaf primordia. We show that it is the direction of the phyllotactic spiral that determines the side of the leaf these asymmetries fall on. We empirically confirm the presence of an asymmetric auxin response using a DR5 reporter and observe morphological asymmetries in young leaf primordia. Notably, these morphological asymmetries persist in mature leaves, and we observe left-right asymmetries in the superficially bilaterally symmetric leaves of tomato (Solanum lycopersicum) and Arabidopsis thaliana that are consistent with modeled predictions. We further demonstrate that auxin application to a single side of a leaf primordium is sufficient to recapitulate the asymmetries we observe. Our results provide a framework to study a previously overlooked developmental axis and provide insights into the developmental constraints imposed upon leaf morphology by auxin-dependent phyllotactic patterning. [ABSTRACT FROM AUTHOR]

The genus Asparagus is unusual in producing axillary, determinate organs called cladodes, which may take on either a flattened or cylindrical form. Here, we investigated the evolution of cladodes to elucidate the mechanisms at play in the diversification of shoot morphology. Our observations of Asparagus asparagoides , which has leaf-like cladodes, showed that its cladodes are anatomically and developmentally similar to leaves but differ in the adaxial/abaxial polarity of the vasculature. In addition to the expression of an ortholog of KNAT1 , orthologous genes that are normally expressed in leaves, ASYMMETRIC LEAVES1 and HD-ZIPIII , were found to be expressed in cladode primordia in a leaf-like manner. The cylindrical cladodes of Asparagus   officinalis showed largely similar expression patterns but showed evidence of being genetically abaxialized. These results provide evidence that cladodes are modified axillary shoots, suggest that the co-option of preexisting gene networks involved in leaf development transferred the leaf-like form to axillary shoots, and imply that altered expression of leaf polarity genes led to the evolution of cylindrical cladodes in the A. officinalis clade. [ABSTRACT FROM AUTHOR]

During leaf development in flowering plants, adaxial (upper) and abaxial (lower) side–specific genes are responsible for blade outgrowth, which takes places predominantly in the lateral direction, and for margin development as well as differentiation of adaxial and abaxial tissues. However, the underlying mechanisms are poorly understood. Here, we show that two WUSCHEL-RELATED HOMEOBOX (WOX) genes, PRESSED FLOWER (PRS)/ WOX3 and WOX1 , encoding homeobox transcription factors, act in blade outgrowth and margin development downstream of adaxial/abaxial polarity establishment. The expression of PRS and WOX1 defines a hitherto undescribed middle domain, including two middle mesophyll layers and the margin, as a center that organizes the outgrowth of leaf blades. The expression of PRS and WOX1 is repressed in the abaxial leaf domain by the abaxial-specific transcription factor KANADI. Furthermore, PRS and WOX1 coordinate adaxial/abaxial patterning together with adaxial- and abaxial-specific genes. Our data suggest a model of blade outgrowth and adaxial/abaxial patterning via the middle domain–specific WOX genes in Arabidopsis thaliana leaves. [ABSTRACT FROM AUTHOR]

Medicago truncatula is a legume species belonging to the inverted repeat lacking clade (IRLC) with trifoliolate compound leaves. However, the regulatory mechanisms underlying development of trifoliolate leaves in legumes remain largely unknown. Here, we report isolation and characterization of fused compound leaf1 (fcl1) mutants of M. truncatula. Phenotypic analysis suggests that FCL1 plays a positive role in boundary separation and proximal-distal axis development of compound leaves. Map-based cloning indicates that FCL1 encodes a class M KNOX protein that harbors the MEINOX domain but lacks the homeodomain. Yeast two-hybrid assays show that FCL1 interacts with a subset of Arabidopsis thaliana BEL1-like proteins with slightly different substrate specificities from the Arabidopsis homolog KNATM-B. Double mutant analyses with M. truncatula single leaflet1 (sgl1) and palmate-like pentafoliata1 (palm1) leaf mutants show that fcl1 is epistatic to palm1 and sgl1 is epistatic to fcl1 in terms of leaf complexity and that SGL1 and FCL1 act additively and are required for petiole development. Previous studies have shown that the canonical KNOX proteins are not involved in compound leaf development in IRLC legumes. The identification of FCL1 supports the role of a truncated KNOX protein in compound leaf development in M. truncatula. [ABSTRACT FROM AUTHOR]

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana   tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [ rpl22 ], rpl23 , rpl32 , ribosomal protein of the small subunit3 [ rps3 ], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36 , yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δ rps15 plants showed only a mild phenotype, Δ rpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway. [ABSTRACT FROM AUTHOR]

Compound leaf development requires highly regulated cell proliferation, differentiation, and expansion patterns. We identified loss-of-function alleles at the SMOOTH LEAF MARGIN1 (SLM1) locus in Medicago truncatula , a model legume species with trifoliate adult leaves. SLM1 encodes an auxin efflux carrier protein and is the ortholog of Arabidopsis thaliana PIN-FORMED1 (PIN1). Auxin distribution is impaired in the slm1 mutant, resulting in pleiotropic phenotypes in different organs. The most striking change in slm1 is the increase in the number of terminal leaflets and a simultaneous reduction in the number of lateral leaflets, accompanied by reduced expression of SINGLE LEAFLET1 (SGL1), an ortholog of LEAFY. Characterization of the mutant indicates that distinct developmental domains exist in the formation of terminal and lateral leaflets. In contrast with the pinnate compound leaves in the wild type, the slm1 sgl1 double mutant shows nonpeltately palmate leaves, suggesting that the terminal leaflet primordium in M. truncatula has a unique developmental mechanism. Further investigations on the development of leaf serrations reveal different ontogenies between distal serration and marginal serration formation as well as between serration and leaflet formation. These data suggest that regulation of the elaboration of compound leaves and serrations is context dependent and tightly correlated with the auxin/SLM1 module in M. truncatula. [ABSTRACT FROM AUTHOR]

Plants contain β-amylase–like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains—also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis -regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain's glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic β-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis -regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic β-amylases, do not influence their transcription factor function. [ABSTRACT FROM AUTHOR]

Leaf senescence is an essential developmental process that impacts dramatically on crop yields and involves altered regulation of thousands of genes and many metabolic and signaling pathways, resulting in major changes in the leaf. The regulation of senescence is complex, and although senescence regulatory genes have been characterized, there is little information on how these function in the global control of the process. We used microarray analysis to obtain a high-resolution time-course profile of gene expression during development of a single leaf over a 3-week period to senescence. A complex experimental design approach and a combination of methods were used to extract high-quality replicated data and to identify differentially expressed genes. The multiple time points enable the use of highly informative clustering to reveal distinct time points at which signaling and metabolic pathways change. Analysis of motif enrichment, as well as comparison of transcription factor (TF) families showing altered expression over the time course, identify clear groups of TFs active at different stages of leaf development and senescence. These data enable connection of metabolic processes, signaling pathways, and specific TF activity, which will underpin the development of network models to elucidate the process of senescence. [ABSTRACT FROM AUTHOR]

CUP-SHAPED COTYLEDON2 (CUC2) and the interacting microRNA miR164 regulate leaf margin dissection. Here, we further investigate the evolution and the specific roles of the CUC1 to CUC3 genes during Arabidopsis thaliana leaf serration. We show that CUC2 is essential for dissecting the leaves of a wide range of lobed/serrated Arabidopsis lines. Inactivation of CUC3 leads to a partial suppression of the serrations, indicating a role for this gene in leaf shaping. Morphometric analysis of leaf development and genetic analysis provide evidence for different temporal contributions of CUC2 and CUC3. Chimeric constructs mixing CUC regulatory sequences with different coding sequences reveal both redundant and specific roles for the three CUC genes that could be traced back to changes in their expression pattern or protein activity. In particular, we show that CUC1 triggers the formation of leaflets when ectopically expressed instead of CUC2 in the developing leaves. These divergent fates of the CUC1 and CUC2 genes after their formation by the duplication of a common ancestor is consistent with the signature of positive selection detected on the ancestral branch to CUC1. Combining experimental observations with the retraced origin of the CUC genes in the Brassicales, we propose an evolutionary scenario for the CUC genes. [ABSTRACT FROM AUTHOR]

C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C4 photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C4 differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g. Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C4 specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database. [ABSTRACT FROM AUTHOR]

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations. [ABSTRACT FROM AUTHOR]

Leaf shape diversity relies on transient morphogenetic activity in leaf margins. However, how this morphogenetic capacity is maintained is still poorly understood. Here, we uncover a role for the hormone cytokinin (CK) in the regulation of morphogenetic activity of compound leaves in tomato (Solanum lycopersicum). Manipulation of CK levels led to alterations in leaf complexity and revealed a unique potential for prolonged growth and morphogenesis in tomato leaves. We further demonstrate that the effect of CK on leaf complexity depends on proper localization of auxin signaling. Genetic analysis showed that reduction of CK levels suppresses the effect of Knotted1 like homeobox (KNOXI) proteins on leaf shape and that CK can substitute for KNOXI activity at the leaf margin, suggesting that CK mediates the activity of KNOXI proteins in the regulation of leaf shape. These results imply that CK regulates flexible leaf patterning by dynamic interaction with additional hormones and transcription factors. [ABSTRACT FROM AUTHOR]

A leaf develops from a few cells that grow, divide, and differentiate to form a complex organ that is precisely positioned relative to its neighbors. How cells communicate to achieve such coordinated growth and development is the focus of this review. We discuss (1) how the stem cells within the shoot meristem gain competence to form organs, (2) what determines the positioning and initiation of new organs, and (3) how the new organ attains its characteristic shape and polarity. Special emphasis is given to the recent integration of mathematics and physics in the study of leaf development. [ABSTRACT FROM AUTHOR]

Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3 -derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development. [ABSTRACT FROM AUTHOR]

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development. [ABSTRACT FROM AUTHOR]

Development of the flattened laminar structure in plant leaves requires highly regulated cell division and expansion patterns. Although tight regulation of these processes is essential during leaf development, leaf shape is highly diverse across the plant kingdom, implying that patterning of growth must be amenable to evolutionary change. Here, we describe the molecular identification of the classical tomato (Solanum lycopersicum) mutant lyrate, which is impaired in outgrowth of leaflet primodia and laminar tissues during compound leaf development. We found that the lyrate phenotype results from a loss-of-function mutation of the tomato JAGGED homolog, a well-described positive regulator of cell division in lateral organs. We demonstrate that LYRATE coordinates lateral outgrowth in the compound leaves of tomato by interacting with both the KNOX and auxin transcriptional networks and suggest that evolutionary changes in LYRATE expression may contribute to the fundamental difference between compound and simple leaves. [ABSTRACT FROM AUTHOR]

Class 1 KNOTTED1-LIKE HOMEOBOX (KNOXI) genes encode transcription factors that are expressed in the shoot apical meristem (SAM) and are essential for SAM maintenance. In some species with compound leaves, including tomato (Solanum lycopersicum), KNOXI genes are also expressed during leaf development and affect leaf morphology. To dissect the role of KNOXI proteins in leaf patterning, we expressed in tomato leaves a fusion of the tomato KNOXI gene Tkn2 with a sequence encoding a repressor domain, expected to repress common targets of tomato KNOXI proteins. This resulted in the formation of small, narrow, and simple leaves due to accelerated differentiation. Overexpression of the wild-type form of Tkn1 or Tkn2 in young leaves also resulted in narrow and simple leaves, but in this case, leaf development was blocked at the initiation stage. Expression of Tkn1 or Tkn2 during a series of spatial and temporal windows in leaf development identified leaf initiation and primary morphogenesis as specific developmental contexts at which the tomato leaf is responsive to KNOXI activity. Arabidopsis thaliana leaves responded to overexpression of Arabidopsis or tomato KNOXI genes during the morphogenetic stage but were largely insensitive to their overexpression during leaf initiation. These results imply that KNOXI proteins act at specific stages within the compound-leaf development program to delay maturation and enable leaflet formation, rather than set the compound leaf route. [ABSTRACT FROM AUTHOR]

Leaf development has been monitored chiefly by following anatomical markers. Analysis of transcriptome dynamics during leaf maturation revealed multiple expression patterns that rise or fall with age or that display age-specific peaks. These were used to formulate a digital differentiation index (DDI) based on a set of selected markers with informative expression during leaf ontogeny. The leaf-based DDI reliably predicted the developmental state of leaf samples from diverse sources and was independent of mitotic cell division transcripts or propensity of specific cell types. When calibrated by informative root markers, the same algorithm accurately diagnosed dissected root samples. We used the DDI to characterize plants with reduced activities of multiple CINCINNATA (CIN)-TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) growth regulators. These plants had giant curled leaves made up of small cells with abnormal shape, low DDI scores, and low expression of mitosis markers, depicting the primary role of CIN-TCPs as promoters of differentiation. Delayed activity of several CIN-TCPs resulted in abnormally large but flat leaves with regular cells. The application of DDI has therefore portrayed the CIN-TCPs as heterochronic regulators that permit the development of a flexible and robust leaf form through an ordered and protracted maturation schedule. [ABSTRACT FROM AUTHOR]

In higher plants, plasmodesmata (PD) are major conduits for cell-cell communication. Primary PD are laid down at cytokinesis, while secondary PD arise during wall extension. During leaf development, the basal cell walls of trichomes extend radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the postcytokinetic wall, simple PD were distributed randomly. As the wall extended, PD became twinned at the cell periphery. Additional secondary pores were inserted at right angles to these, giving rise to pit fields composed of several paired PD. During wall extension, the number of PD increased fivefold due to the insertion of secondary PD. Our data are consistent with a model in which a subset of the original primary PD pores function as templates for the insertion of new secondary PD, spatially fixing the position of future pit fields. Many of the new PD shared the same wall collar as the original PD pore, suggesting that new PD pores may arise by fissions of existing PD progenitors. Different models of secondary PD formation are discussed. Our data are supported by a computational model, Plasmodesmap, which accurately simulates the formation of radial pit fields during cell wall extension based on the occurrence of multiple PD twinning events in the cell wall. The model predicts PD distributions with striking resemblance to those seen on fractured wall faces. [ABSTRACT FROM AUTHOR]

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLOI/FY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLOILFY gene took over this role in the IRLC. [ABSTRACT FROM AUTHOR]