Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana, we showed that the cell-cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.

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.

Despite its relevance for agricultural production, environmental stress-induced growth inhibition, which is responsible for significant yield reductions, is only poorly understood. Here, we investigated the molecular mechanisms underlying cell cycle inhibition in young proliferating leaves of the model plant Arabidopsis thaliana when subjected to mild osmotic stress. A detailed cellular analysis demonstrated that as soon as osmotic stress is sensed, cell cycle progression rapidly arrests, but cells are kept in a latent ambivalent state allowing a quick recovery (pause). Remarkably, cell cycle arrest coincides with an increase in 1-aminocyclopropane-1-carboxylate levels and the activation of ethylene signaling. Our work showed that ethylene acts on cell cycle progression via inhibition of cyclin-dependent kinase A activity independently of EIN3 transcriptional control. When the stress persists, cells exit the mitotic cell cycle and initiate the differentiation process (stop). This stop is reflected by early endoreduplication onset, in a process independent of ethylene. Nonetheless, the potential to partially recover the decreased cell numbers remains due to the activity of meristemoids. Together, these data present a conceptual framework to understand how environmental stress reduces plant growth.

When subjected to stress, plants reprogram their growth by largely unknown mechanisms. To provide insights into this process, the growth of Arabidopsis (Arabidopsis thaliana) leaves that develop under mild osmotic stress was studied. Early during leaf development, cell number and size were reduced by stress, but growth was remarkably adaptable, as division and expansion rates were identical to controls within a few days of leaf initiation. To investigate the molecular basis of the observed adaptability, leaves with only proliferating, exclusively expanding, and mature cells were analyzed by transcriptomics and targeted metabolomics. The stress response measured in growing and mature leaves was largely distinct; several hundred transcripts and multiple metabolites responded exclusively in the proliferating and/or expanding leaves. Only a few genes were differentially expressed across the three stages. Data analysis showed that proliferation and expansion were regulated by common regulatory circuits, involving ethylene and gibberellins but not abscisic acid. The role of ethylene was supported by the analysis of ethylene-insensitive mutants. Exclusively in proliferating cells, stress induced genes of the so-called "mitochondrial dysfunction regulon," comprising alternative oxidase. Up-regulation for eight of these genes was confirmed with promoter:beta-glucuronidase reporter lines. Furthermore, mitochondria of stress-treated dividing cells were morphologically distinct from control ones, and growth of plants overexpressing the alternative oxidase gene was more tolerant to osmotic and drought stresses. Taken together, our data underline the value of analyzing stress responses in development and demonstrate the importance of mitochondrial respiration for sustaining cell proliferation under osmotic stress conditions.

SUMMARYIntracellular invasion of root cells is required for the establishment of successful endosymbioses in legumes of both arbuscular mycorrhizal (AM) fungi and rhizobial bacteria. In both interactions a requirement for successful entry is the activation of a common signalling pathway that includes five genes required to generate calcium oscillations and two genes required for the perception of the calcium response. Recently, it has been discovered that in Medicago truncatula, the Vapyrin (VPY) gene is essential for the establishment of the arbuscular mycorrhizal symbiosis. Here, we show by analyses of mutants that the same gene is also required for rhizobial colonization and nodulation. VPY encodes a protein featuring a Major Sperm Protein domain, typically featured on proteins involved in membrane trafficking and biogenesis, and a series of ankyrin repeats. Plants mutated in this gene have abnormal rhizobial infection threads and fewer nodules, and in the case of interactions with AM fungi, epidermal penetration defects and aborted arbuscule formation. Calcium spiking in root hairs in response to supplied Nod factors is intact in the vpy-1 mutant. This, and the elevation of VPY transcripts upon application of Nod factors which we show to be dependent on NFP, DMI1, and DMI3, indicates that VPY acts downstream of the common signalling pathway.

The establishment of the photosynthetic apparatus during chloroplast development creates a high demand for iron as a redox metal. However, iron in too high quantities becomes toxic to the plant, thus plants have evolved a complex network of iron uptake and regulation mechanisms. Here, we examined whether four of the subgroup Ib basic helix-loop-helix transcription factors (bHLH38, bHLH39, bHLH100, bHLH101), previously implicated in iron homeostasis in roots, also play a role in regulating iron metabolism in developing leaves. These transcription factor genes were strongly up-regulated during the transition from cell proliferation to expansion, and thus sink-source transition, in young developing leaves of Arabidopsis thaliana. The four subgroup Ib bHLH genes also showed reduced expression levels in developing leaves of plants treated with norflurazon, indicating their expression was tightly linked to the onset of photosynthetic activity in young leaves. In addition, we provide evidence for a mechanism whereby the transcriptional regulators SAC51 and TCP20 antagonistically regulate the expression of these four subgroup Ib bHLH genes. A loss-of-function mutant analysis also revealed that single mutants of bHLH38, bHLH39, bHLH100, and bHLH101 developed smaller rosettes than wild-type plants in soil. When grown in agar plates with reduced iron concentration, triple bhlh39 bhlh100 bhlh101 mutant plants were smaller than wild-type plants. However, measurements of the iron content in single and multiple subgroup Ib bHLH genes, as well as transcript profiling of iron response genes during early leaf development, do not support a role for bHLH38, bHLH39, bHLH100, and bHLH101 in iron homeostasis during early leaf development.

BackgroundExisting statistical methods for tiling array transcriptome data either focus on transcript discovery in one biological or experimental condition or on the detection of differential expression between two conditions. Increasingly often, however, biologists are interested in time-course studies, studies with more than two conditions or even multiple-factor studies. As these studies are currently analyzed with the traditional microarray analysis techniques, they do not exploit the genome-wide nature of tiling array data to its full potential.ResultsWe present an R Bioconductor package, , which implements a wavelet-based model for analyzing transcriptome data and extends it towards more complex experimental designs. With the user is able to discover (1) group-wise expressed regions, (2) differentially expressed regions between any two groups in single-factor studies and in (3) multifactorial designs. Moreover, for time-course experiments it is also possible to detect (4) linear time effects and (5) a circadian rhythm of transcripts. By considering the expression values of the individual tiling probes as a function of genomic position, effect regions can be detected regardless of existing annotation. Three case studies with different experimental set-ups illustrate the use and the flexibility of the model-based transcriptome analysis.ConclusionsThe package provides the user with a convenient tool for the analysis of tiling array trancriptome data for a multitude of experimental set-ups. Regardless of the study design, the probe-wise analysis allows for the detection of transcriptional effects in both exonic, intronic and intergenic regions, without prior consultation of existing annotation.