Use of Synchrotron Phase-Sensitive Imaging for the Investigation of Magnetopriming and Solar UV-Exclusion Impact on Soybean (Glycine max) Leaves.

The combined response of exclusion of solar ultraviolet radiation (UV-A+B and UV-B) and static magnetic field (SMF) pre-treatment of 200 mT for 1 h were studied on soybean (Glycine max) leaves using synchrotron imaging. The seeds of soybean with and without SMF pre-treatment were sown in nursery bags kept in iron meshes where UV-A+B (280-400 nm) and UV-B (280-315 nm) from solar radiation were filtered through a polyester filters. Two controls were planned, one with polythene filter controls (FC)- which allows all the UV (280-400 nm); the other control had no filter used (open control-OC). Midrib regions of the intact third trifoliate leaves were imaged using the phase-contrast imaging technique at BL-4, Indus-2 synchrotron radiation source. The solar UV exclusion results suggest that ambient UV caused a reduction in leaf growth which ultimately reduced the photosynthesis in soybean seedlings, while SMF treatment caused enhancement of leaf growth along with photosynthesis even under the presence of ambient UV-B stress. The width of midrib and second-order veins, length of the second-order veins, leaf vein density, and the density of third-order veins obtained from the quantitative image analysis showed an enhancement in the leaves of plants that emerged from SMF pre-treated seeds as compared to untreated ones grown in open control and filter control conditions (in the presence of ambient UV stress). SMF pre-treated seeds along with UV-A+B and UV-B exclusion also showed significant enhancements in leaf parameters as compared to the UV excluded untreated leaves. Our results suggested that SMF-pretreatment of seeds diminishes the ambient UV-induced adverse effects on soybean.

Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase.

When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2',7'-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase-mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity.

Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri)vascular tissue induced by sunlight and paraquat.

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (>1100µmolm-2s-1 of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H2O2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H2O2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H2O2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H2O2 in signaling were discussed.

Anatomical constraints to C4 evolution: light harvesting capacity in the bundle sheath.

In C4 photosynthesis CO2 assimilation and reduction are typically coordinated across mesophyll (M) and bundle sheath (BS) cells, respectively. This system consequently requires sufficient light to reach BS to generate enough ATP to allow ribulose-1,5-bisphosphate (RuBP) regeneration in BS. Leaf anatomy influences BS light penetration and therefore constrains C4 cycle functionality. Using an absorption scattering model (coded in Excel, and freely downloadable) we simulate light penetration profiles and rates of ATP production in BS across the C3 , C3 -C4 and C4 anatomical continua. We present a trade-off for light absorption between BS pigment concentration and space allocation. C3 BS anatomy limits light absorption and benefits little from high pigment concentrations. Unpigmented BS extensions increase BS light penetration. C4 and C3 -C4 anatomies have the potential to generate sufficient ATP in the BS, whereas typical C3 anatomy does not, except some C3 taxa closely related to C4 groups. Insufficient volume of BS, relative to M, will hamper a C4 cycle via insufficient BS light absorption. Thus, BS ATP production and RuBP regeneration, coupled with increased BS investments, allow greater operational plasticity. We propose that larger BS in C3 lineages may be co-opted for C3 -C4 and C4 biochemistry requirements.

In Planta Response of Arabidopsis to Photothermal Impact Mediated by Gold Nanoparticles.

Biological responses to photothermal effects of gold nanoparticles (GNPs) have been demonstrated and employed for various applications in diverse systems except for one important class - plants. Here, the uptake of GNPs through Arabidopsis thaliana roots and translocation to leaves are reported. Successful plasmonic nanobubble generation and acoustic signal detection in planta is demonstrated. Furthermore, Arabidopsis leaves harboring GNPs and exposed to continuous laser or noncoherent light show elevated temperatures across the leaf surface and induced expression of heat-shock regulated genes. Overall, these results demonstrate that Arabidopsis can readily take up GNPs through the roots and translocate the particles to leaf tissues. Once within leaves, GNPs can act as photothermal agents for on-demand remote activation of localized biological processes in plants.

Light-induced plasticity in leaf hydraulics, venation, anatomy, and gas exchange in ecologically diverse Hawaiian lobeliads.

Leaf hydraulic conductance (Kleaf ) quantifies the capacity of a leaf to transport liquid water and is a major constraint on light-saturated stomatal conductance (gs ) and photosynthetic rate (Amax ). Few studies have tested the plasticity of Kleaf and anatomy across growth light environments. These provided conflicting results. The Hawaiian lobeliads are an excellent system to examine plasticity, given the striking diversity in the light regimes they occupy, and their correspondingly wide range of Amax , allowing maximal carbon gain for success in given environments. We measured Kleaf , Amax , gs and leaf anatomical and structural traits, focusing on six species of lobeliads grown in a common garden under two irradiances (300/800 µmol photons m(-2)  s(-1) ). We tested hypotheses for light-induced plasticity in each trait based on expectations from optimality. Kleaf , Amax , and gs differed strongly among species. Sun/shade plasticity was observed in Kleaf , Amax, and numerous traits relating to lamina and xylem anatomy, venation, and composition, but gs was not plastic with growth irradiance. Species native to higher irradiance showed greater hydraulic plasticity. Our results demonstrate that a wide set of leaf hydraulic, stomatal, photosynthetic, anatomical, and structural traits tend to shift together during plasticity and adaptation to diverse light regimes, optimizing performance from low to high irradiance.

Was low CO2 a driving force of C4 evolution: Arabidopsis responses to long-term low CO2 stress.

The responses of long-term growth of plants under elevated CO2 have been studied extensively. Comparatively, the responses of plants to subambient CO2 concentrations have not been well studied. This study aims to investigate the responses of the model C3 plant, Arabidopsis thaliana, to low CO2 at the molecular level. Results showed that low CO2 dramatically decreased biomass productivity, together with delayed flowering and increased stomatal density. Furthermore, alteration of thylakoid stacking in both bundle sheath and mesophyll cells, upregulation of PEPC and PEPC-K together with altered expression of a number of regulators known involved in photosynthesis development were observed. These responses to low CO2 are discussed with regard to the fitness of C3 plants under low CO2. This work also briefly discusses the relevance of the data to C4 photosynthesis evolution.

Fluorescence F 0 of photosystems II and I in developing C3 and C 4 leaves, and implications on regulation of excitation balance.

This work addresses the question of occurrence and function of photosystem II (PSII) in bundle sheath (BS) cells of leaves possessing NADP-malic enzyme-type C4 photosynthesis (Zea mays). Although no requirement for PSII activity in the BS has been established, several component proteins of PSII have been detected in BS cells of developing maize leaves exhibiting O2-insensitive photosynthesis. We used the basal fluorescence emissions of PSI (F 0I) and PSII (F 0II) as quantitative indicators of the respective relative photosystem densities. Chl fluorescence induction was measured simultaneously at 680 and 750 nm. In mature leaves, the F m(680)/F 0(680) ratio was 10.5 but less in immature leaves. We propose that the lower ratio was caused by the presence of a distinct non-variable component, F c, emitting at 680 and 750 nm. After F c was subtracted, the fluorescence of PSI (F 0I) was detected as a non-variable component at 750 nm and was undetectably low at 680 nm. Contents of Chls a and b were measured in addition to Chl fluorescence. The Chl b/(a + b) was relatively stable in developing sunflower leaves (0.25-0.26), but in maize it increased from 0.09 to 0.21 with leaf tissue age. In sunflower, the F 0I/(F 0I + F 0II) was 0.39 ± 0.01 independent of leaf age, but in maize, this parameter was 0.65 in young tissue of very low Chl content (20-50 mg m(-2)) falling to a stable level of 0.53 ± 0.01 at Chl contents >100 mg m(-2). The values of F 0I/(F 0I + F 0II) showed that in sunflower, excitation was partitioned between PSII and PSI in a ratio of 2:1, but the same ratio was 1:1 in the C4 plant. The latter is consistent with a PSII:PSI ratio of 2:1 in maize mesophyll cells and PSI only in BS cells (2:1:1 distribution). We suggest, moreover, that redox mediation of Chl synthesis, rather than protein accumulation, regulates photosystem assembly to ensure optimum excitation balance between functional PSII and PSI. Indeed, the apparent necessity for two Chls (a and b) may reside in their targeted functions in influencing accumulation of PSI and PSII, respectively, as opposed to their spectral differences.

From proto-Kranz to C4 Kranz: building the bridge to C4 photosynthesis.

In this review, we examine how the specialized "Kranz" anatomy of C4 photosynthesis evolved from C3 ancestors. Kranz anatomy refers to the wreath-like structural traits that compartmentalize the biochemistry of C4 photosynthesis and enables the concentration of CO2 around Rubisco. A simplified version of Kranz anatomy is also present in the species that utilize C2 photosynthesis, where a photorespiratory glycine shuttle concentrates CO2 into an inner bundle-sheath-like compartment surrounding the vascular tissue. C2 Kranz is considered to be an intermediate stage in the evolutionary development of C4 Kranz, based on the intermediate branching position of C2 species in 14 evolutionary lineages of C4 photosynthesis. In the best-supported model of C4 evolution, Kranz anatomy in C2 species evolved from C3 ancestors with enlarged bundle sheath cells and high vein density. Four independent lineages have been identified where C3 sister species of C2 plants exhibit an increase in organelle numbers in the bundle sheath and enlarged bundle sheath cells. Notably, in all of these species, there is a pronounced shift of mitochondria to the inner bundle sheath wall, forming an incipient version of the C2 type of Kranz anatomy. This incipient version of C2 Kranz anatomy is termed proto-Kranz, and is proposed to scavenge photorespiratory CO2. By doing so, it may provide fitness benefits in hot environments, and thus represent a critical first stage of the evolution of both the C2 and C4 forms of Kranz anatomy.

Acclimation of C4 metabolism to low light in mature maize leaves could limit energetic losses during progressive shading in a crop canopy.

C4 plants have a biochemical carbon-concentrating mechanism that increases CO2 concentration around Rubisco in the bundle sheath. Under low light, the activity of the carbon-concentrating mechanism generally decreases, associated with an increase in leakiness (ϕ), the ratio of CO2 retrodiffusing from the bundle sheath relative to C4 carboxylation. This increase in ϕ had been theoretically associated with a decrease in biochemical operating efficiency (expressed as ATP cost of gross assimilation, ATP/GA) under low light and, because a proportion of canopy photosynthesis is carried out by shaded leaves, potential productivity losses at field scale. Maize plants were grown under light regimes representing the cycle that leaves undergo in the canopy, whereby younger leaves initially developed under high light and were then re-acclimated to low light (600 to 100 µE·m(-2)·s(-1) photosynthetically active radiation) for 3 weeks. Following re-acclimation, leaves reduced rates of light-respiration and reached a status of lower ϕ, effectively optimizing the limited ATP resources available under low photosynthetically active radiation. Direct estimates of respiration in the light, and ATP production rate, allowed an empirical estimate of ATP production rate relative to gross assimilation to be derived. These values were compared to modelled ATP/GA which was predicted using leakiness as the sole proxy for ATP/GA, and, using a novel comprehensive biochemical model, showing that irrespective of whether leaves are acclimated to very low or high light intensity, the biochemical efficiency of the C4 cycle does not decrease at low photosynthetically active radiation.

Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 regulates xylem development and growth by a conserved mechanism that modulates hormone signaling.

Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-ß-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways.

Acclimation to humidity modifies the link between leaf size and the density of veins and stomata.

The coordination of veins and stomata during leaf acclimation to sun and shade can be facilitated by differential epidermal cell expansion so large leaves with low vein and stomatal densities grow in shade, effectively balancing liquid- and vapour-phase conductances. As the difference in vapour pressure between leaf and atmosphere (VPD) determines transpiration at any given stomatal density, we predict that plants grown under high VPD will modify the balance between veins and stomata to accommodate greater maximum transpiration. Thus, we examined the developmental responses of these traits to contrasting VPD in a woody angiosperm (Toona ciliata M. Roem.) and tested whether the relationship between them was altered. High VPD leaves were one-third the size of low VPD leaves with only marginally greater vein and stomatal density. Transpirational homeostasis was thus maintained by reducing stomatal conductance. VPD acclimation changed leaf size by modifying cell number. Hence, plasticity in vein and stomatal density appears to be generated by plasticity in cell size rather than cell number. Thus, VPD affects cell number and leaf size without changing the relationship between liquid- and vapour-phase conductances. This results in inefficient acclimation to VPD as stomata remain partially closed under high VPD.

Acclimation to low light by C4 maize: implications for bundle sheath leakiness.

C4 plants have a biochemical carbon concentrating mechanism (CCM) that increases CO2 concentration around ribulose bisphosphate carboxylase oxygenase (Rubisco) in the bundle sheath (BS). Under limiting light, the activity of the CCM generally decreases, causing an increase in leakiness, (Φ), the ratio of CO2 retrodiffusing from the BS relative to C4 carboxylation processes. Maize plants were grown under high and low light regimes (respectively HL, 600 versus LL, 100 µE m(-2) s(-1) ). Short-term acclimation of Φ was compared from isotopic discrimination (Δ), gas exchange and photochemistry. Direct measurement of respiration in the light, and ATP production rate (JATP ), allowed us use a novel approach to derive Φ, compared with the conventional fitting of measured and predicted Δ. HL grown plants responded to decreasing light intensities with the well-documented increase in Φ. Conversely, LL plants showed a constant Φ, which has not been observed previously. We explain the pattern by two contrasting acclimation strategies: HL plants maintained a high CCM activity at LL, resulting in high CO2 overcycling and increased Φ; LL plants acclimated by down-regulating the CCM, effectively optimizing scarce ATP supply. This surprising plasticity may limit the impact of Φ-dependent carbon losses in leaves becoming shaded within developing canopies.

How do leaf veins influence the worldwide leaf economic spectrum? Review and synthesis.

Leaf vein traits are implicated in the determination of gas exchange rates and plant performance. These traits are increasingly considered as causal factors affecting the 'leaf economic spectrum' (LES), which includes the light-saturated rate of photosynthesis, dark respiration, foliar nitrogen concentration, leaf dry mass per area (LMA) and leaf longevity. This article reviews the support for two contrasting hypotheses regarding a key vein trait, vein length per unit leaf area (VLA). Recently, Blonder et al. (2011, 2013) proposed that vein traits, including VLA, can be described as the 'origin' of the LES by structurally determining LMA and leaf thickness, and thereby vein traits would predict LES traits according to specific equations. Careful re-examination of leaf anatomy, published datasets, and a newly compiled global database for diverse species did not support the 'vein origin' hypothesis, and moreover showed that the apparent power of those equations to predict LES traits arose from circularity. This review provides a 'flux trait network' hypothesis for the effects of vein traits on the LES and on plant performance, based on a synthesis of the previous literature. According to this hypothesis, VLA, while virtually independent of LMA, strongly influences hydraulic conductance, and thus stomatal conductance and photosynthetic rate. We also review (i) the specific physiological roles of VLA; (ii) the role of leaf major veins in influencing LES traits; and (iii) the role of VLA in determining photosynthetic rate per leaf dry mass and plant relative growth rate. A clear understanding of leaf vein traits provides a new perspective on plant function independently of the LES and can enhance the ability to explain and predict whole plant performance under dynamic conditions, with applications towards breeding improved crop varieties.

The evolution of angiosperm lianescence without vessels--climbing mode and wood structure-function in Tasmannia cordata (Winteraceae).

• The lack of extant lianescent vessel-less seed plants supports a hypothesis that liana evolution requires large-diameter xylem conduits. Here, we demonstrate an unusual example of a lianoid vessel-less angiosperm, Tasmannia cordata (Winteraceae), from New Guinea. • Wood mechanical, hydraulic and structural measurements were used to determine how T. cordata climbs and to test for ecophysiological shifts related to liana evolution vs 13 free-standing congeners. • The tracheid-based wood of T. cordata furnished low hydraulic capacity compared with that of vessel-bearing lianas. In comparison with most nonclimbing relatives, T. cordata possessed lower photosynthetic rates and leaf and stem hydraulic capacities. However, T. cordata exhibited a two- to five-fold greater wood elastic modulus than its relatives. • Tasmannia cordata provides an unusual example of angiosperm liana evolution uncoupled from xylem conduit gigantism, as well as high plasticity and cell type diversity in vascular development. Because T. cordata lacks vessels, our results suggest that a key limitation for a vessel-less liana is that strong and low hydraulically conductive wood is required to meet the mechanical demands of lianescence.

DBB1a, involved in gibberellin homeostasis, functions as a negative regulator of blue light-mediated hypocotyl elongation in Arabidopsis.

Double B-box 1a (DBB1a) belongs to the zinc-finger family proteins in Arabidopsis thaliana. Transcriptional analysis uncovered that the DBB1a gene expression was blue light-dependently regulated, and the transcript level of DBB1a in cry1cry2 was decreased but not in phyAphyB compared to wild type under blue light conditions. Transgenic plants containing pDBB1a:GUS (ß-glucuronidase) displayed GUS activity in the vascular system of leaves and petioles. Green fluorescent protein (GFP)-fused DDB1a (DBB1a-GFP) protein was found in the nucleus in transient transformation assays with onion epidermal cells as well as in stable transgenic Arabidopsis plants. To investigate the function of DBB1a, we generated DBB1a over-expressing and under-expressing transgenic Arabidopsis plants. Analysis of hypocotyl growth of these lines indicated that DBB1a promoted hypocotyl elongation under blue light condition. The phenotype of transgenic plants with DBB1a over-expression could be impaired by a gibberellin (GA)-biosynthesis inhibitor. Moreover, the expression analysis of GA metabolic and catabolic genes in DBB1a transgenic lines indicated that the DBB1a suppressed GA2-oxidase1 (GA2ox1) and GA2-oxidase8 (GA2ox8) expression, but induced GA3ß-hydroxygenase1 (GA3ox1) and GA20-oxidase1 (GA20ox1) expression under blue light. Taken together, we concluded that DBB1a promotes hypocotyl elongation under blue light condition through an increase in bioactive GA levels in Arabidopsis.

Amino acid compositional shifts during streptophyte transitions to terrestrial habitats.

Across the streptophyte lineage, which includes charophycean algae and embryophytic plants, there have been at least four independent transitions to the terrestrial habitat. One of these involved the evolution of embryophytes (bryophytes and tracheophytes) from a charophycean ancestor, while others involved the earliest branching lineages, containing the monotypic genera Mesostigma and Chlorokybus, and within the Klebsormidiales and Zygnematales lineages. To overcome heat, water stress, and increased exposure to ultraviolet radiation, which must have accompanied these transitions, adaptive mechanisms would have been required. During periods of dehydration and/or desiccation, proteomes struggle to maintain adequate cytoplasmic solute concentrations. The increased usage of charged amino acids (DEHKR) may be one way of maintaining protein hydration, while increased use of aromatic residues (FHWY) protects proteins and nucleic acids by absorbing damaging UV, with both groups of residues thought to be important for the stabilization of protein structures. To test these hypotheses we examined amino acid sequences of orthologous proteins representing both mitochondrion- and plastid-encoded proteomes across streptophytic lineages. We compared relative differences within categories of amino acid residues and found consistent patterns of amino acid compositional fluxuation in extra-membranous regions that correspond with episodes of terrestrialization: positive change in usage frequency for residues with charged side-chains, and aromatic residues of the light-capturing chloroplast proteomes. We also found a general decrease in the usage frequency of hydrophobic, aliphatic, and small residues. These results suggest that amino acid compositional shifts in extra-membrane regions of plastid and mitochondrial proteins may represent biochemical adaptations that allowed green plants to colonize the land.