Borate and phosphite treatments of potato plants (Solanum tuberosum L.) as a proof of concept to reinforce the cell wall structure and reduce starch digestibility.

Potatoes are one of the main sources of carbohydrates in human diet, however they have a high glycaemic index (GI). Hence, developing new agricultural and industrial strategies to produce low GI potatoes represents a health priority to prevent obesity and related diseases. In this work, we investigated whether treatments of potato plants with elicitors of plant defence responses can lead to a reduction of tuber starch availability and digestibility, through the induction of cell wall remodelling and stiffening. Treatments with phosphites (KPhi) and borate were performed, as they are known to activate plant defence responses that cause modifications in the architecture and composition of the plant cell wall. Data of suberin autofluorescence demonstrated that potato plants grown in a nutrition medium supplemented with KPhi and borate produced tubers with a thicker periderm, while pectin staining demonstrated that KPhi treatment induced a reinforcement of the wall of storage parenchyma cells. Both compounds elicited the production of H2O2, which is usually involved in cell-wall remodelling and stiffening reactions while only KPhi caused an increase of the total content of phenolic compounds. A two-phase digestion in vitro assay showed that treatment with KPhi determined a significant decrease of the starch hydrolysis rate in potato tubers. This work highlights the ability of cell wall architecture in modulating starch accessibility to digestive enzymes, paving the way for new agronomic practices to produce low GI index potatoes.

LncRNA TCONS_00021861 is functionally associated with drought tolerance in rice (Oryza sativa L.) via competing endogenous RNA regulation.

BACKGROUND: Water deficit is an abiotic stress that retards plant growth and destabilizes crop production. Long non coding RNAs (lncRNAs) are a class of non-coding endogenous RNAs that participate in diverse cellular processes and stress responses in plants. lncRNAs could function as competing endogenous RNAs (ceRNA) and represent a novel layer of gene regulation. However, the regulatory mechanism of lncRNAs as ceRNA in drought stress response is yet unclear. RESULTS: In this study, we performed transcriptome-wide identification of drought-responsive lncRNAs in rice. Thereafter, we constructed a lncRNA-mediated ceRNA network by analyzing competing relationships between mRNAs and lncRNAs based on ceRNA hypothesis. A drought responsive ceRNA network with 40 lncRNAs, 23 miRNAs and 103 mRNAs was obtained. Network analysis revealed TCONS_00021861/miR528-3p/YUCCA7 regulatory axis as a hub involved in drought response. The miRNA-target expression and interaction were validated by RT-qPCR and RLM-5'RACE. TCONS_00021861 showed significant positive correlation (r = 0.7102) with YUCCA7 and negative correlation with miR528-3p (r = -0.7483). Overexpression of TCONS_00021861 attenuated the repression of miR528-3p on YUCCA7, leading to increased IAA (Indole-3-acetic acid) content and auxin overproduction phenotypes. CONCLUSIONS: TCONS_00021861 could regulate YUCCA7 by sponging miR528-3p, which in turn activates IAA biosynthetic pathway and confer resistance to drought stress. Our findings provide a new perspective of the regulatory roles of lncRNAs as ceRNAs in drought resistance of rice.

Starch Granules in Arabidopsis thaliana Mesophyll and Guard Cells Show Similar Morphology but Differences in Size and Number.

Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

TGD5 is required for normal morphogenesis of non-mesophyll plastids, but not mesophyll chloroplasts, in Arabidopsis.

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.

Coleataenia prionitis, a C4-like species in the Poaceae.

C4-like plants represent the penultimate stage of evolution from C3 to C4 plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C4 plant, its leaf anatomy and gas exchange traits suggest that it may be a C4-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C4 Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C4-like intermediate plants rather than C4 plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO2. These data demonstrate that C. prionitis is not a true C4 plant but should be considered as a C4-like plant.

Complexity of Brassica oleracea-Alternaria brassicicola Susceptible Interaction Reveals Downregulation of Photosynthesis at Ultrastructural, Transcriptional, and Physiological Levels.

Black spot disease, caused by Alternaria brassicicola in Brassica species, is one of the most devastating diseases all over the world, especially since there is no known fully resistant Brassica cultivar. In this study, the visualization of black spot disease development on Brassica oleracea var. capitata f. alba (white cabbage) leaves and subsequent ultrastructural, molecular and physiological investigations were conducted. Inter- and intracellular hyphae growth within leaf tissues led to the loss of host cell integrity and various levels of organelle disintegration. Severe symptoms of chloroplast damage included the degeneration of chloroplast envelope and grana, and the loss of electron denseness by stroma at the advanced stage of infection. Transcriptional profiling of infected leaves revealed that photosynthesis was the most negatively regulated biological process. However, in infected leaves, chlorophyll and carotenoid content did not decrease until 48 hpi, and several chlorophyll a fluorescence parameters, such as photosystem II quantum yield (Fv/Fm), non-photochemical quenching (NPQ), or plant vitality parameter (Rdf) decreased significantly at 24 and 48 hpi compared to control leaves. Our results indicate that the initial stages of interaction between B. oleracea and A. brassicicola are not uniform within an inoculation site and show a complexity of host responses and fungal attempts to overcome host cell defense mechanisms. The downregulation of photosynthesis at the early stage of this susceptible interaction suggests that it may be a part of a host defense strategy, or, alternatively, that chloroplasts are targets for the unknown virulence factor(s) of A. brassicicola. However, the observed decrease of photosynthetic efficiency at the later stages of infection is a result of the fungus-induced necrotic lesion expansion.

Seasonal movement of chloroplasts in mesophyll cells of two Picea species.

Cold acclimation in evergreen conifers of temperate zone is associated with seasonal structural changes of mesophyll cells. Photoprotective reactions include the movement of chloroplasts from summer position when they are located along the cell walls to winter arrangement with their aggregation in one part of the cell. Special spatial arrangement of mesophyll in Picea species with chloroplasts located along the two opposite cell walls causes the very specific pattern of chloroplast movement. To reveal the intracellular apparatus involved in the seasonal organelle position changes, 3D reconstruction of mesophyll cell structure was applied. Two Picea species, P. obovata and P. pungens, from two geographic regions were studied in a 3-year course. The involvement of small transparent vacuoles in the development of cytoplasmic strands penetrating through the central vacuole and connecting two opposite lateral sides of the cell was shown. The nucleus and cytoplasmic organelles including chloroplasts move inwards the strand forming the cytoplasmic conglomerate enclosed by the vacuole at the cell center. Two Picea species have distinct differences in spatial organization of winter mesophyll cells and in structural events leading to its formation. Analysis of Picea species from two geographic regions over 3 years of monitoring reveals dependence of seasonal organelle movement on the dynamics of temperature decline in autumn.

Elevated CO2-induced changes in mesophyll conductance and anatomical traits in wild type and carbohydrate-metabolism mutants of Arabidopsis.

Decreases in photosynthetic rate, stomatal conductance (gs), and mesophyll conductance (gm) are often observed under elevated CO2 conditions. However, which anatomical and/or physiological factors contribute to the decrease in gm is not fully understood. Arabidopsis thaliana wild-type and carbon-metabolism mutants (gwd1, pgm1, and cfbp1) with different accumulation patterns of non-structural carbohydrates were grown at ambient (400 ppm) and elevated (800 ppm) CO2. Anatomical and physiological traits of leaves were measured to investigate factors causing the changes in gm and in the mesophyll resistance (expressed as the reciprocal of mesophyll conductance per unit chloroplast surface area facing to intercellular space, Sc/gm). When grown at elevated CO2, all the lines showed increases in cell wall mass, cell wall thickness, and starch content, but not in leaf thickness. gm measured at 800 ppm CO2 was significantly lower than at 400 ppm CO2 in all the lines. Changes in Sc/gm were associated with thicker cell walls rather than with excess starch content. The results indicate that the changes in gm and Sc/gm that occur in response to elevated CO2 are independent of non-structural carbohydrates, and the cell wall represents a greater limitation factor for gm than starch.

Loss of LHCI system affects LHCII re-distribution between thylakoid domains upon state transitions.

LHCI, the peripheral antenna system of Photosystem I, includes four light-harvesting proteins (Lhca1-Lhca4) in higher plants, all of which are devoid in the Arabidopsis thaliana knock-out mutant ΔLhca. PSI absorption cross-section was reduced in the mutant, thus affecting the redox balance of the photosynthetic electron chain and resulting in a more reduced PQ with respect to the wild type. ΔLhca plants developed compensatory response by enhancing LHCII binding to PSI. However, the amplitude of state transitions, as measured from changes of chlorophyll fluorescence in vivo, was unexpectedly low than the high level of PSI-LHCII supercomplex established. In order to elucidate the reasons for discrepancy, we further analyzed state transition in ΔLhca plants. The STN7 kinase was fully active in the mutant as judged from up-regulation of LHCII phosphorylation in state II. Instead, the lateral heterogeneity of thylakoids was affected by lack of LHCI, with LHCII being enriched in stroma membranes with respect to the wild type. Re-distribution of this complex affected the overall fluorescence yield of thylakoids already in state I and minimized changes in RT fluorescence yield when LHCII did connect to PSI reaction center. We conclude that interpretation of chlorophyll fluorescence analysis of state transitions becomes problematic when applied to mutants whose thylakoid architecture is significantly modified with respect to the wild type.

Three-dimensional ultrastructure of chloroplast pockets formed under salinity stress.

We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three-dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam-scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam-scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.

Biochemical and transcriptomic analyses of drought stress responses of LY1306 tobacco strain.

This study aimed to investigate drought resistance of the LY1306 tobacco strain. Seedlings of tobacco strains LY1306, ZhongYan 100 (ZY100) and Hong Hua Da Jin Yuan (HHDJY) were treated with polyethylene glycol (PEG)-6000 to induce osmotic stress. As validation, water-deficit-induced drought treatments, including mild drought (MD; watering 1.5 L/week) and severe drought (SD, without watering) were carried out. Changes in cell morphology, leaf water potential, antioxidant enzyme activity, as well as contents of malondialdehyde (MDA) and proline were determined for each treatment. Transcriptome sequencing was performed for the seedlings treated with 15% PEG-6000. No obvious changes were observed in morphology of LY1306 and ZY100 under osmotic or drought stress; whereas, visible wilting was observed in HHDJY. Superoxide dismutase and peroxidase activities of LY1036 and ZY100 under osmotic stress were significantly higher than those of HHDJY. Under SD, the MDA content of LY1306 was significantly lower and the proline content of LY1306 was significantly higher than that of HHDJY. Differential genes between LY1306, ZY100 and HHDJY were enriched in functions about alpha-linolenic acid, and arginine and proline metabolisms. LY1306 could increase its antioxidant enzyme activities and proline accumulation in response to drought stress, probably by regulating drought resistance-related pathways and genes.

Mesophyll cell ultrastructure of wheat leaves etiolated by lead and selenium.

The ultrastructure of mesophyll cells was studied in leaves of the Triticum aestivum L. cv. "Trizo" seedlings after two weeks of growth on soil contaminated by Pb and/or Se. The soil treatments: control; (Pb1) 50mgkg-1; (Pb2) 100mgkg-1; (Se1) 0.4mgkg-1; (Se2) 0.8mgkg-1; (Pb1+Se1); (Pb1+Se2); (P2+Se1); and (Pb2+Se2) were used. Light and other conditions were optimal for plant growth. The (Se1)-plants showed enhanced growth and biomass production; (Pb1+Se1)-plants did not lag behind the controls, though O2 evolution decreased; chlorophyll content did not differ statistically in these treatments. Other treatments led to statistically significant growth suppression, chlorophyll content reduction, inhibition of photosynthesis, stress development tested by H2O2 and leaf etiolation at the end of 14-days experiment. The tops of etiolated leaves remained green, while the main leaf parts were visually white. Plastids in mesophyll cells of etiolated parts of leaves were mainly represented by etioplasts and an insignificant amount of degraded chloroplasts. Other cellular organelles remained intact in most mesophyll cells of the plants, except (Pb2+Se2)-plants. Ruptured tonoplast and etioplast envelope, swelled cytoplasm and mitochondria, and electron transparent matrix of gialoplasm were observed in the mesophyll cells at (Pb2+Se2)-treatment, that caused maximal inhibition of plant growth. The results indicate that Pb and Se effects on growth of wheat leaves are likely to target meristem in which the development of proplastids to chloroplasts under the light is determined by chlorophyll biosynthesis. Antagonistic effect of low concentration of Se and Pb in combination may retard etiolation process.

Phloem Loading through Plasmodesmata: A Biophysical Analysis.

In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model-including local and global transport and kinetics of polymerization-for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export.

Intracellular position of mitochondria in mesophyll cells differs between C3 and C4 grasses.

In C3 plants, part of the CO2 fixed during photosynthesis in chloroplasts is released from mitochondria during photorespiration by decarboxylation of glycine via glycine decarboxylase (GDC), thereby reducing photosynthetic efficiency. The apparent positioning of most mitochondria in the interior (vacuole side of chloroplasts) of mesophyll cells in C3 grasses would increase the efficiency of refixation of CO2 released from mitochondria by ribulose 1,5-bisphosphate carboxylase/​oxygenase (Rubisco) in chloroplasts. Therefore, in mesophyll cells of C4 grasses, which lack both GDC and Rubisco, the mitochondria ought not to be positioned the same way as in C3 mesophyll cells. To test this hypothesis, we investigated the intracellular position of mitochondria in mesophyll cells of 14 C4 grasses of different C4 subtypes and subfamilies (Chloridoideae, Micrairoideae, and Panicoideae) and a C3-C4 intermediate grass, Steinchisma hians, under an electron microscope. In C4 mesophyll cells, most mitochondria were positioned adjacent to the cell wall, which clearly differs from the positioning in C3 mesophyll cells. In S. hians mesophyll cells, the positioning was similar to that in C3 cells. These results suggest that the mitochondrial positioning in C4 mesophyll cells reflects the absence of both GDC and Rubisco in the mesophyll cells and the high activity of phosphoenolpyruvate carboxylase. In contrast, the relationship between the mitochondrial positioning and enzyme distribution in S. hians is complex, but the positioning may be related to the capture of respiratory CO2 by Rubisco. Our study provides new possible insight into the physiological role of mitochondrial positioning in photosynthetic cells.

Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM.

Background and Aims: Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized. In this study, a whole rice mesophyll cell was observed using a focused ion beam scanning electron microscope (FIB-SEM), which provides many serial sections automatically, rapidly and correctly, thereby enabling 3-D cell structure reconstruction. Methods: Rice leaf blades were fixed chemically using the method for conventional TEM observation, embedded in resin and subsequently set in the FIB-SEM chamber. Specimen blocks were sectioned transversely using the FIB, and block-face images were captured using the SEM. The sectioning and imaging were repeated overnight for 200-500 slices (each 50 nm thick). The resultant large-volume image stacks ( x = 25 µm, y = 25 µm, z = 10-25 µm) contained one or two whole mesophyll cells. The 3-D models of whole mesophyll cells were reconstructed using image processing software. Key Results: The reconstructed cell models were discoid shaped with several lobes around the cell periphery. The cell shape increased the surface area, and the ratio of surface area to volume was twice that of a cylinder having the same volume. The chloroplasts occupied half the cell volume and spread as sheets along the cell lobes, covering most of the inner cell surface, with adjacent chloroplasts in close contact with each other. Conclusions: Cellular and sub-cellular ultrastructures of a whole mesophyll cell in a rice leaf blade are demonstrated three-dimensionally using a FIB-SEM. The 3-D models and numerical information support the hypothesis that rice mesophyll cells enhance their CO 2 absorption with increased cell surface and sheet-shaped chloroplasts.

Comparison of cellular responses to Xanthomonas perforans infection between resistant and susceptible tomato accessions.

Bacterial spot of tomato (Solanum lycopersicum L.) caused by several Xanthomonas species is one of the most destructive diseases. Genes regulating the hypersensitive resistance and field resistance to X. perforans race T3 have been intensively investigated over the last decade. However, a comparative analysis of cellular responses to the pathogen in susceptible and resistant hosts has not been completed, which prevents the detailed understanding of the interactions between the pathogen and tomato plants. In this study, the characteristics of lesions, stomata, and pathogen colonization in hypersensitive response (HR) PI 128216, field-resistant PI 114490, and susceptible OH 88119 tomato plants after inoculation with green fluorescent protein-labeled X. perforans race T3 bacteria were investigated. Significant differences in developmental processes and the micromorphology of spot lesions among three tomato lines were observed. Our results suggested that the faster lesion development in OH 88119 plants compared with that of the other two lines was associated with a greater increase in the stomatal apertures over a longer period following bacterial inoculation. The depth of bacterial colonization and pathogen density inside infected leaves in OH 88119 were also significantly different from that of resistant tomato plants. Determination of the ultrastructural responses to X. perforans among three tomato lines revealed that cell wall defense response was the main difference between resistant and susceptible tomato lines. These results may provide fundamental information for understanding the cellular and molecular mechanisms regulating tomato responses to X. perforans race T3.

Characterization and fine-mapping of a novel premature leaf senescence mutant yellow leaf and dwarf 1 in rice.

Leaves are the main organs in which photosynthates are produced. Leaf senescence facilitates the translocation of photosynthates and nutrients from source to sink, which is important for plant development and especially for crop yield. However, the molecular mechanism of leaf senescence is unknown. Here, we identified a mutant, yellow leaf and dwarf 1 (yld1), which exhibited decreased plant height and premature leaf senescence. Nitroblue tetrazolium and diamiobenzidine staining analyses revealed that the concentrations of reactive oxygen species were higher in yld1 leaves than in wild type leaves. The photosynthetic pigment contents were significantly decreased in yld1. The yld1 chloroplasts had collapsed and were filled with abnormal starch granules. Combining bulk segregant and MutMap gene mapping approaches, the mutation responsible for the yld1 phenotype was mapped to a 7.3 Mb centromeric region, and three non-synonymous single nucleotide polymorphisms located in three novel genes were identified in this region. The expression patterns of the three candidate genes indicated that LOC_Os06g29380 had the most potential for functional verification. Plant hormone measurements showed that salicylic acid was highly accumulated in yld1 leaves when compared with wild type leaves, and yld1 was more sensitive to salicylic acid than wild type. This work lays the foundation for understanding the molecular regulatory mechanism of leaf senescence, and may reveal new connections among the molecular pathways related to leaf senescence, starch metabolism and salicylic acid signaling.

Leaf anatomy mediates coordination of leaf hydraulic conductance and mesophyll conductance to CO2 in Oryza.

Leaf hydraulic conductance (Kleaf ) and mesophyll conductance (gm ) both represent major constraints to photosynthetic rate (A), and previous studies have suggested that Kleaf and gm is correlated in leaves. However, there is scarce empirical information about their correlation. In this study, Kleaf , leaf hydraulic conductance inside xylem (Kx ), leaf hydraulic conductance outside xylem (Kox ), A, stomatal conductance (gs ), gm , and anatomical and structural leaf traits in 11 Oryza genotypes were investigated to elucidate the correlation of H2 O and CO2 diffusion inside leaves. All of the leaf functional and anatomical traits varied significantly among genotypes. Kleaf was not correlated with the maximum theoretical stomatal conductance calculated from stomatal dimensions (gsmax ), and neither gs nor gsmax were correlated with Kx . Moreover, Kox was linearly correlated with gm and both were closely related to mesophyll structural traits. These results suggest that Kleaf and gm are related to leaf anatomical and structural features, which may explain the mechanism for correlation between gm and Kleaf .

RNA processing body (P-body) dynamics in mesophyll protoplasts re-initiating cell division.

The ability of plants to regenerate lies in the capacity of differentiated cells to reprogram and re-enter the cell cycle. Reprogramming of cells requires changes in chromatin organisation and gene expression. However, there has been less focus on changes at the post transcription level. We have investigated P-bodies, sites of post transcriptional gene regulation, in plant cell reprogramming in cultured mesophyll protoplasts; by using a YFP-VARICOSE (YFP-VCSc) translational fusion. We showed an early increase in P-body number and volume, followed by a decline, then a subsequent continued increase in P-body number and volume as cell division was initiated and cell proliferation continued. We infer that plant P-bodies have a role to play in reprogramming the mature cell and re-initiating the cell division cycle. The timing of the first phase is consistent with the degredation of messages no longer required, as the cell transits to the division state, and may also be linked to the stress response associated with division induction in cultured cells. The subsequent increase in P-body formation, with partitioning to the daughter cells during the division process, suggests a role in the cell cycle and its re-initiation in daughter cells. P-bodies were shown to be mobile in the cytoplasm and show actin-based motility which facilitates their post-transcriptional role and partitioning to daughter cells.