Project description:Plants have evolved sophisticated cellular and molecular mechanisms to acclimate to adverse environmental stresses. Here, we report that acute heat stress induces excessive cell expansion, reaching up to 13,000 μm2, in Arabidopsis thaliana. The heat-induced cell expansion progressed along with programmed cell death. Autophagy was responsible for excessive cell expansion, as evidenced by autophagic structures in the expanded cells as well as failure of heat-induced cell expansion in autophagy-related 5-1 (atg5-1) and atg7-2 mutants. We also reveal that the transcription factor ETHYLENE RESPONSE FACTOR115 (ERF115), which is activated upon neighboring cell death, is involved in heat-induced cell expansion. ERF115 accumulation in response to heat possibly activated the expression of several ATGs by directly binding to gene promoters. In agreement, the erf115 phytochrome a signal transduction1-2 (pat1-2) double mutant exhibited reduced cell expansion under heat stress with diminished autophagic activity. Overall, our findings indicate that heat-stress-induced cell death triggers autophagy-mediated cell expansion in plant leaves, which may compensate for the death of nearby cells.
Project description:Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.
Project description:For the detailed and sensitive detection of the responses to a specific treatment it is important to perform tissue or cell type specific analyses, but this is not easily achievable for the different leaf tissues. Here we developed a method termed MeSelect to effectively separate leaf epidermis, vascular and mesophyll cells basically without contamination from other tissues. The high yield of the MeSelect method allowed for tissue specific high-throughput proteome analyses after inhibition of the proteasome with Syringolin A and the affinity enrichment of polyubiquitylated proteins in epidermis, mesophyll and vasculature.
Project description:For the detailed and sensitive detection of the responses to a specific treatment it is important to perform tissue or cell type specific analyses, but this is not easily achievable for the different leaf tissues. Here we developed a method termed MeSelect to effectively separate leaf epidermis, vascular and mesophyll cells basically without contamination from other tissues. The high yield of the MeSelect method allowed for tissue specific high-throughput proteome analyses after inhibition of the proteasome with Syringolin A and the affinity enrichment of polyubiquitylated proteins in epidermis, mesophyll and vasculature.
Project description:Understanding the physiological responses of crops to drought is important for ensuring sustained crop productivity under climate change, which is expected to exacerbate the frequency and intensity of periods of drought. Drought responses involve multiple traits, and the correlations between these traits are poorly understood. Using a variety of techniques, we estimated the changes in gas exchange, leaf hydraulic conductance, and leaf turgor in rice (Oryza sativa) in response to both short- and long-term soil drought. We performed a photosynthetic limitation analysis to quantify the contributions of each limiting factor to the resultant overall decrease in photosynthesis during drought. Biomass, leaf area, and leaf width significantly decreased during the 2-week drought treatment, but leaf mass per area and leaf vein density increased. Light-saturated photosynthetic rate declined dramatically during soil drought, mainly due to the decrease in stomatal conductance (gs) and mesophyll conductance (gm). Stomatal modeling suggested that the decline in leaf hydraulic conductance explained most of the decrease in stomatal closure during the drought treatment, and may also trigger the drought-related decrease of stomatal conductance and mesophyll conductance. The results of this study provide insight into the regulation of carbon assimilation under drought conditions.
Project description:Leaf function is influenced by leaf structure, which is itself related not only to the spatial arrangement of constituent mesophyll cells, but also their size and shape. In this study, we used confocal microscopy to image leaves of Triticum genotypes varying in ploidy level to extract 3D information on individual mesophyll cell size and geometry. Combined with X-ray Computed Tomography and gas exchange analysis, the effect of changes in wheat mesophyll cell geometry upon leaf structure and function were investigated. Mesophyll cell size and shape were found to have changed during the course of wheat evolution. An unexpected linear relationship between mesophyll cell surface area and volume was discovered, suggesting anisotropic scaling of mesophyll cell geometry with increasing ploidy. Altered mesophyll cell size and shape were demonstrated to be associated with changes in mesophyll tissue architecture. Under experimental growth conditions, CO2 assimilation did not vary with ploidy, but stomatal conductance was lower in hexaploid plants, conferring a greater instantaneous water-use efficiency. We propose that as wheat mesophyll cells have become larger with increased ploidy, this has been accompanied by changes in cell geometry and packing which limit water loss while maintaining carbon assimilation.
Project description:BackgroundDespite the wide spread application of confocal and multiphoton laser scanning microscopy in plant biology, leaf phenotype assessment still relies on two-dimensional imaging with a limited appreciation of the cells' structural context and an inherent inaccuracy of cell measurements. Here, a successful procedure for the three-dimensional imaging and analysis of plant leaves is presented.ResultsThe procedure was developed based on a range of developmental stages, from leaf initiation to senescence, of soil-grown Arabidopsis thaliana (L.) Heynh. Rigorous clearing of tissues, made possible by enhanced leaf permeability to clearing agents, allowed the optical sectioning of the entire leaf thickness by both confocal and multiphoton microscopy. The superior image quality, in resolution and contrast, obtained by the latter technique enabled the three-dimensional visualisation of leaf morphology at the individual cell level, cell segmentation and the construction of structural models. Image analysis macros were developed to measure leaf thickness and tissue proportions, as well as to determine for the epidermis and all layers of mesophyll tissue, cell density, volume, length and width. For mesophyll tissue, the proportion of intercellular spaces and the surface areas of cells were also estimated. The performance of the procedure was demonstrated for the expanding 6th leaf of the Arabidopsis rosette. Furthermore, it was proven to be effective for leaves of another dicotyledon, apple (Malus domestica Borkh.), which has a very different cellular organisation.ConclusionsThe pipeline for the three-dimensional imaging and analysis of plant leaves provides the means to include variables on internal tissues in leaf growth studies and the assessment of leaf phenotypes. It also allows the visualisation and quantification of alterations in leaf structure alongside changes in leaf functioning observed under environmental constraints. Data obtained using this procedure can further be integrated in leaf development and functioning models.
Project description:In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.
Project description:With growing concerns over global warming, cultivating heat-tolerant crops has become paramount to prepare for the anticipated warmer climate. Cassava (Manihot esculenta Crantz), a vital tropical crop, demonstrates exceptional growth and productivity under high-temperature (HT) conditions. Yet, studies elucidating HT resistance mechanisms in cassava, particularly within vascular tissues, are rare. We dissected the leaf mid-vein from leaf, and did the comparative transcriptome profiling between mid-vein and leaf to figure out the cassava vasculature HT resistance molecular mechanism. Anatomical microscopy revealed that cassava leaf veins predominantly consisted of vasculature. A thermal imaging analysis indicated that cassava experienced elevated temperatures, coinciding with a reduction in photosynthesis. Transcriptome sequencing produced clean reads in total of 89.17G. Using Venn enrichment, there were 65 differentially expressed genes (DEGs) and 93 DEGs had been found highly specifically expressed in leaf and mid-vein. Further investigation disclosed that leaves enhanced pyruvate synthesis as a strategy to withstand high temperatures, while mid-veins fortified themselves by bolstering lignin synthesis by comprehensive GO and KEGG analysis of DEGs. The identified genes in these metabolic pathways were corroborated through quantity PCR (QPCR), with results aligning with the transcriptomic data. To verify the expression localization of DEGs, we used in situ hybridization experiments to identify the expression of MeCCoAMT(caffeoyl-coenzyme A-3-O-methyltransferase) in the lignin synthesis pathway in cassava leaf veins xylem. These findings unravel the disparate thermotolerance mechanisms exhibited by cassava leaves and mid-veins, offering insights that could potentially inform strategies for enhancing thermotolerance in other crops.