Hydraulic design of leaves: insights from rehydration kinetics

We examined the leaf hydraulic design in 10 species based on their rehydration kinetics. In all cases, a biphasic response described the temporal pattern of water uptake, with time constants of approximately 30 to 800 s and approximately 800 to 8000 s. The time constants of the fast phase were significantly shorter in the six angiosperms (30 to 110 s) compared with the two single-veined conifer species (>400 s) examined, while the two multi-veined gymnosperm species, Gnetum gnemon and Ginkgo biloba, had time constants for the fast phase of approximately 150 s. Among angiosperm species, the fast phase constituted 50-90% of the total water absorbed, whereas in gymnosperms 70-90% of the water uptake could be assigned to the slow phase. In the four gymnosperms, the relative water uptake corresponding to the fast phase matched to a good degree the relative volume of the venation and bundle sheath extension; whereas in the angiosperm species, the relatively larger water influx during the fast phase was similar in relative volume to the combined venation, bundle sheath extension, epidermis and (in four species) the spongy mesophyll. This suggests a general trend from a design in which the epidermis is weakly connected to the veins (all four gymnosperms), to a design with good hydraulic connection between epidermis and veins that largely bypasses the mesophyll (four of six angiosperms), to a design in which almost the entire leaf appears to function as a single pool.     

An allosteric rheostat in HIV-1 gp120 reduces CCR5 stoichiometry required for membrane fusion and overcomes diverse entry limitations

Binding of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120 to the CCR5 co-receptor reduces constraints on the metastable transmembrane subunit gp41, thereby enabling gp41 refolding, fusion of viral and cellular membranes, and infection. We previously isolated adapted HIV-1_JRCSF variants that more efficiently use mutant CCR5s, including CCR5(Δ18) lacking the important tyrosine sulfate-containing amino terminus. Effects of mutant CCR5 concentrations on HIV-1 infectivities were highly cooperative, implying that several may be required. However, because wild-type CCR5 efficiently mediates infections at trace concentrations that were difficult to measure accurately, analyses of its cooperativity were not feasible. New HIV-1_JRCSF variants efficiently use CCR5(HHMH), a chimera containing murine extracellular loop 2. The adapted virus induces large syncytia in cells containing either wild-type or mutant CCR5s and has multiple gp120 mutations that occurred independently in CCR5(Δ18)-adapted virus. Accordingly, these variants interchangeably use CCR5(HHMH) or CCR5(Δ18). Additional analyses strongly support a novel energetic model for allosteric proteins, implying that the adaptive mutations reduce quaternary constraints holding gp41, thus lowering the activation energy barrier for membrane fusion without affecting bonds to specific CCR5 sites. In accordance with this mechanism, highly adapted HIV-1s require only one associated CCR5(HHMH), whereas poorly adapted viruses require several. However, because they are allosteric ensembles, complexes with additional co-receptors fuse more rapidly and efficiently than minimal ones. Similarly, wild-type HIV-1_JRCSF is highly adapted to wild-type CCR5 and minimally requires one. The adaptive mutations cause resistances to diverse entry inhibitors and cluster appropriately in the gp120 trimer interface overlying gp41. We conclude that membrane fusion complexes are allosteric machines with an ensemble of compositions, and that HIV-1 adapts to entry limitations by gp120 mutations that reduce its allosteric hold on gp41. These results provide an important foundation for understanding the mechanisms that control membrane fusion and HIV-1's facile adaptability.     

Differential inhibition of photosynthesis under drought stress in <Emphasis Type="Italic">Flaveria</Emphasis> species with different degrees of development of the C<Subscript>4</Subscript> syndrome

The effect of drought stress (DS) on photosynthesis and photosynthesis-related enzyme activities was investigated in F. pringlei (C₃), F. floridana (C₃–C₄), F. brownii (C₄-like), and F. trinervia (C₄) species. Stomatal closure was observed in all species, probably being the main cause for the decline in photosynthesis in the C₃ species under ambient conditions. In vitro ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and stromal fructose 1,6-bisphosphatase (sFBP) activities were sufficient to interpret the net photosynthetic rates (P _N), but, from the decreases in P _N values under high CO₂ (C _a = 700 μmol mol^{− 1}) it is concluded that a decrease in the in vivo rate of the RuBPCO reaction may be an additional limiting factor under DS in the C₃ species. The observed decline in the photosynthesis capacity of the C₃–C₄ species is suggested to be associated both to in vivo decreases of RuBPCO activity and of the RuBP regeneration rate. The decline of the maximum P _N observed in the C₄-like species under DS was probably attributed to a decrease in maximum RuBPCO activity and/or to decrease of enzyme substrate (RuBP or PEP) regeneration rates. In the C₄ species, the decline of both in vivo photosynthesis and photosynthetic capacity could be due to in vivo inhibition of the phosphoenolpyruvate carboxylase (PEPC) by a twofold increase of the malate concentration observed in mesophyll cell extracts from DS plants.     

Molecular properties of two proteins homologous to PduO-type ATP:cob(I)alamin adenosyltransferase from Sulfolobus tokodaii

In the thermophilic archaeon Sulfolobus tokodaii, there are two genes homologous to PduO-type ATP:cob(I)alamin adenosyltransferase, ST1454 and ST2180. To address the structure and function of these two sequence-related proteins from one organism, we prepared them by using the Escherichia coli expression system and analyzed them by immunoblotting, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy, circular dichroism spectrometry, ATP:cobalamin adenosyltransferase assay, and X-ray crystallography. Immunoblotting and matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy analyses showed that both these proteins are expressed in S. tokodaii cells as soluble proteins and are spontaneously digested at the N-terminal region. ATP:cob(I)alamin adenosyltransferase activity was detected for ST1454 but not for ST2180. ST2180 reduced the concentration of cob(I)alamin, suggesting that ST2180 might recognize cob(I)alamin as a ligand. The secondary structure of ST1454 was retained even in 7 M guanidine hydrochroride, whereas that of ST2180 was melted in 4.5 M guanidine hydrochloride. The X-ray crystal structural analysis revealed that the proteins shared a common structure: a trimer of five-helix bundles with a clockwise kink. There is a pocket surrounded by highly conserved residues, in which a polypropylene glycol 400 in the crystal structure of ST1454 was captured, suggesting that it is an active site. Structural comparison between these two proteins showed the difference in the number of ion pairs around the proposed active site. On the basis of these results, we propose that ST1454 and ST2180 have related but distinct functions.     

13CO2/12CO2 exchange fluxes in a clamp‐on leaf cuvette: disentangling artefacts and flux components

Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO₂ concentration (up to ±250 μmol mol^?1) and δ¹³C_CO2 (34‰), between a clamp‐on leaf cuvette (LI‐6400) and surrounding air, to assess (1) leak coefficients for CO₂, ¹²CO₂, and ¹³CO₂ with the empty cuvette and with intact leaves of Holcus lanatus (C₃) or Sorghum bicolor (C₄) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for ¹²CO₂ and ¹³CO₂, but ～8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%).     

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild‐type and leaf vein mutants

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (K_leaf), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col‐0) and four vein mutants (dot3‐111 and dot3‐134, and cvp1‐3 and cvp2‐1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean K_leaf. Across all genotypes, there was a strong correlation of K_leaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence K_leaf, indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing K_leaf.     

Divergent evolution of a bifunctional de novo protein

Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn‐IF, was chosen from a library of binary patterned 4‐helix bundles. Syn‐IF was shown previously to rescue two different auxotrophic strains of E. coli: ΔilvA and Δfes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn‐IF, was evolved for faster rescue of either ΔilvA or Δfes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity.     

热消散法(TDP)在5种竹子蒸腾耗水测定中的适用性评价

为评价热消散法在竹子蒸腾耗水测定中的适用性,利用室内离体竹段注水变压法结合野外整株容器称重法对Granier 公式进行了验证和系数校正,同时观察了5 种竹子(毛竹Phyllostachys edulis、粉单竹Bambusa chungii、青皮竹B. textilis、茶秆竹Arundinaria amabilis、龙头竹B. vulgaris)的茎秆维管束结构。结果表明,茎秆维管束分布不均,维管束发育程度从竹壁外向内逐渐成熟,输水能力也逐渐增强。液流密度(F_d)和液流指数(K)呈幂函数关系,相关系数R²>0.83,说明热消散探针方法能较好地估算竹类的液流密度。用整株容器称重法对推导出的液流密度公式进行校正,校正后的液流密度公式与原始Granier 公式中各系数均不同,尤其是α 值。分别采用校正前后公式计算的竹子日蒸腾量差异显著,尤其是一天中液流高峰时段(午间)的差异最大。因此,只要对热消散探针方法准确验证、校正Granier 原始公式系数,TDP 技术是估计竹类植物水分利用的一种适宜方法。     

叶脉网络系统的构建和系统学意义研究进展

为了解国内外叶脉网络系统的研究状况,综述了基因、激素对叶脉网络系统发育的调控机理,并剖析了叶脉的功能和系统学意义,分析了光、温度、水和外力破坏等环境因子对叶脉密度、叶脉直径等结构性状的影响。同时,综合考量植物碳投入经济权衡,阐明了叶脉网络系统是在遗传控制基础上由环境与碳投入共同调控建成。最后,对植物叶脉网络系统研究中存在的问题与未来发展方向进行了展望。