遮光对香果树幼苗光合特性及叶片解剖结构的影响

设置了全光照(L0)、透光率60%(L1)、透光率25%(L2)、透光率10%(L3)4种光环境,研究了不同遮光处理对香果树幼苗光合作用及叶片解剖结构的影响。结果表明:(1)遮光对香果树幼苗净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(G_s)、气孔限制值(L_s)、胞间CO_2浓度(C_i)、蒸汽压亏缺(V_(pdl))均产生了显著影响。全光和L1处理下Pn和Tr日变化呈明显的双峰曲线,中午出现了明显的"午休",其余处理的变化相对缓和,双峰和"午休"现象不明显;G_s的日变化规律与Pn基本一致;C_i的日变化整体呈先期缓慢降低后期抬升的趋势,与对照相比遮光显著提高了叶片胞间CO_2浓度;Ls和V_(pdl)呈现出先升后降的弧形变化,并且遮光下L_s和V_(pdl)日均值显著低于全光照。一天中P_n和T_r的日均值大小排序为L1L0L2L3。(2)不同光环境下香果树幼苗的光合响应特征明显不同。全光下,香果树幼苗能够维持较高的净光合作用速率而未呈现明显的光抑制,表现出一定的光忍耐和喜光性。L1处理下的最大净光合速率(P_(max))、暗呼吸速率(R_d)和表观量子效率(AQY)显著增加,说明香果树对光强具有一定的可塑性,轻度遮光有利于光合效率提高;强度遮光(L2、L3)导致光补偿点(LCP)和光饱和点(LSP)显著降低,有利于充分利用弱光,但P_(max)、R_d明显下降,光合效率受到抑制。(3)遮光显著影响了香果树叶片的气孔分布。遮光条件下香果树叶片的气孔密度显著减小,气孔器长度、宽度与单个气孔器面积也呈现类似的变化。(4)遮光条件下香果树叶片厚度减小,上表皮增厚,下表/ST增加;维管束内单列导管数目和导管直径均有明显增大,促进了叶片水分传导运输。(5)在育苗生产中,为了适应香果树幼苗生长发育的需求,建议有效辐射强度保持在自然光强的60%以上。     

Light regulation of photosynthetic membrane structure,organization, and function

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light-harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun-adapted species and plants growing under far-red-depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade-adapted species and plants growing under far-red-enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.     

Correlations among leaf structure,drought tolerance and photosynthetic capacity in saplings of Euphorbiaceae from different micro-habitats in a seasonal tropical rainforest

Aims Leaf structural and functional traits have been extensively studied to explain community assembly mechanisms, species distributions, niche differentiations, and even ecosystem services functions. However, these traits are influenced by both environment and phylogeny, showing correlations or trade-offs among them. In this study, we assessed the impacts of leaf structure on drought tolerance and photosynthetic potential, and the trade-off between drought tolerance and photosynthetic capacity, to provide an explanation for species coexistence and the maintenance of high biodiversity in tropical rainforests.
Methods We chose 18 species in the Euphobiaceae family differing in distribution patterns along topographic gradients in a 20 hm² forest dynamics monitoring plot (FDP) in Xishuangbanna. We measured leaf anatomy, leaf water relations characteristics, maximum photosynthetic rate, and dark respiration, and used two different methods—the traditional Pearson correlation and phylogenetic independent contrasts—to analyze the relationships among those traits.
Important findings We found that: 1) species showed convergence in structures and functions within specific habitat; species on ridge or slope had a stronger water loss-tolerance abilities than species in the valley. 2) Correlations among some key traits (specifically, leaf density, water potential at turgor loss point, and water potential at full turgor, etc.) were found among habitats; plants adjusted leaf structure to influence simultaneously plant water loss-tolerance abilities and photosynthetic capability, which may result in a trade-off between drought tolerance (high leaf density, leaf mass per area) and photosynthetic capability (low leaf density, leaf mass per area). 3) The phylogenetic independent contrasts must be used when analyzing correlations among the traits of geneticallyrelated species due to the weakness of traditional Pearson analysis. The ecological niche differentiation to water and light gradients as revealed by the present study provides a potential explanation for the high diversity of the seasonal tropical rainforest.     

热带季雨林不同小生境大戟科植物幼树的叶片结构、耐旱性和光合能力之间的相关性

植物的叶片结构和功能性状受到自身、环境和系统发育的影响。该研究选取西双版纳20 hm²热带雨林动态监测大样地内18种分布格局不同的大戟科植物, 测量了幼树叶片的解剖结构、水分关系特征、最大光合能力和暗呼吸, 主要探讨了叶片结构对植物耐旱性和光合能力的影响, 耐旱性和光合能力之间的权衡关系, 以及环境水分条件对植物功能性状相关性的影响。结果表明: 1)生境内植物表现出一定的结构和功能的趋同性, 分布在山脊和山坡的种比沟谷种具有更强的耐失水能力; 2)去除了系统发育的影响后, 一些关键性状(特别是叶片密度和膨压丧失点时的水势、饱和渗透势等)之间存在跨生境尺度上的相关关系, 植物叶片结构同时影响了植物的耐失水能力和光合能力, 植物叶片自身的结构限制导致了植物的耐旱性(高的叶片密度、比叶质量)和光合能力(低的叶片密度、比叶质量)存在反向进化关系; 3)如果研究的植物类群亲缘关系较近, 传统的Pearson相关分析不能很好地揭示其性状间的相关关系, 因而必须采用系统发育独立对照差作相关分析。大戟科植物的结构和功能在水分梯度和光梯度上的生态位分化也从功能性状的角度为热带季雨林能维持高生物多样性, 保持植物物种长期共存提供了一个可能的解释。     

Phototropic leaf movements and photosynthetic performance in an amphibious fern, Marsilea quadrifolia

Diurnal phototropism has not been reported in ferns. In this study we found that the four leaflets of the amphibious fern Marsilea quadrifolia are capable of adjusting their leaflet angle and leaflet azimuth in response to changes in the position of the sun’s direct beam, exhibiting more diaphototropic movements (orienting the plane of the lamina perpendicular to incident light) in the morning and late afternoon, and more paraphototropic movements (orienting the plane of the lamina parallel to incident light) at noon. In addition, by cutting off the leaflet lamina and covering portions of leaflets with black tape, the junction between the leaflet and petiole was found to be responsible for light reception. Among the light spectrum investigated, blue light was the most effective at inducing diaphototropism. The role of diurnal phototropism in enhancing carbon return and ameliorating photoinhibition was also evaluated. It was concluded that diurnal phototropic leaf movement represents one of the plastic responses enabling this amphibious fern to grow under terrestrial conditions.     

Slow development of leaf photosynthesis in an evergreen broad-leaved tree, Castanopsis sieboldii: relationships between leaf anatomical characteristics and photosynthetic rate

Changes in net photosynthetic rate on a leaf area basis and anatomical properties during leaf development were studied in an evergreen broad‐leaved tree, Castanopsis sieboldii and an annual herb, Phaseolus vulgaris. In C. sieboldii, surface area of mesophyll cells facing the intercellular air spaces on a leaf area basis (S_mes) was already considerable at the time of full leaf area expansion (FLE). However, surface area of chloroplasts facing the intercellular air spaces on a leaf area basis (S_c), and chlorophyll and Rubisco contents on a leaf area basis increased to attain their maximal values 15–40 d after FLE. In contrast, in P. vulgaris, chloroplast number on a leaf area basis, S_c and S_mes at 10 d before FLE were two to three times greater than the steady‐state levels attained at around FLE. In C. sieboldii, the internal CO₂ transfer conductance (g_i) slightly increased for 10 d after FLE but then decreased toward the later stages. Limitation of photosynthesis by g_i was only about 10% at FLE, but then increased to about 30% at around 40 d after FLE. The large limitation after FLE by g_i was probably due to the decrease in CO₂ concentration in the chloroplast caused by the increases in thickness of mesophyll cell walls and in Rubisco content per chloroplast surface area. These results clearly showed that: (1) in C. sieboldii, chloroplast development proceeded more slowly than mesophyll cell expansion and continued well after FLE, whereas in P. vulgaris these processes proceeded synchronously and were completed by FLE; (2) after FLE, photosynthesis in leaves of C. sieboldii was markedly limited by g_i. From these results, it is suggested that, in the evergreen broad‐leaved trees, mechanical protection of mesophyll cells has priority over the efficient CO₂ transfer and quick construction of the chloroplasts.     

Canopy and leaf photosynthetic characteristics and water use efficiency of sweet sorghum under drought stress

The objective of this paper was to examine the relationship between Water Use Efficiency (WUE) at the canopy and leaf levels, to determine soil moisture conditions, which can optimize yield, and WUE of sweet sorghum (Sorghum bicolor (Linn.) Moench), thus providing some theoretical foundation for using marginal land effectively and developing production of sweet sorghum. Three levels of soil moisture conditions were established, and photosynthetic characteristics and yield were measured. The canopy apparent photo-synthetic rate (CAP) and leaf photosynthetic rate (P _N) were reduced gradually with increased drought stress, and the CAP was lower than the P _N under every soil moisture conditions. The P _N had a midday depression phenomenon, but the CAP did not exhibit this midday depression phenomenon under severe drought stress. The linear regression relationship of CAP and P _N was CAP = 1.5945 + 0.1496 P _N. The canopy apparent WUE_C and leaf WUE_L were the highest under moderate drought stress. The first was 5.3 and 5.8 times higher than the WUE_L in mid-July and late August, respectively. The stem fresh biomass yield was 77 tons/ha under moderate drought stress and WUE of aboveground biomass yield (WUE_B) was also the highest. Our results showed that moderate drought stress did not result in a significant reduction in biomass yield but increased WUE significantly.     

Relationship between salt tolerance and photosynthetic machinery performance in citrus

In citrus, salt stress has been related to the build up of chloride ions in plant tissues that affect photosynthesis, growth and yield. We investigated the effects of salt stress on the stability of the photosynthetic machinery with respect to the relative salt tolerance of different citrus genotypes including: Swingle Citrumelo, Carrizo citrange, C35 citrange, Cleopatra mandarin and Forner-Alcaide #5. Under identical salt-stress conditions, Forner-Alcaide #5 and Cleopatra mandarin accumulated less chloride ions in leaves than the other genotypes and showed a better plant performance. Chlorophyll fluorescence parameters indicated severe impairments of photosynthetic activity in salt-sensitive Citrumelo and citranges but Cleopatra and Forner-Alcaide #5 were less affected. In addition, differences in photosynthetic responses between these two moderately tolerant genotypes suggested different strategies to cope with salinity. The high tolerance to salinity shown by Forner-Alcaide #5 can be associated to the ability of keeping an active photosynthetic system at elevated saline conditions whereas the tolerance of Cleopatra was linked to rapid reductions of net photosynthetic rate, stomatal conductance, performance of PSII and photosynthetic efficiency.     

青钱柳叶片解剖结构及黄酮、三萜和多糖的组织化学定位观察

青钱柳〔Cyclocarya paliurus ( Batal.) Iljinskaja〕隶属于胡桃科( Juglandaceae)青钱柳属( Cyclocarya Iljinskaja) ,为中国特有的单种属植物,具有较高的药用、材用和观赏等价值[1];青钱柳叶片中含有多种次生代谢成分,具有多种药理活性[2-4].目前,对于青钱柳次生代谢成分的研究主要集中在提取、分离和纯化及不同种源间次生代谢成分的变化规律等方面,其中,已有研究者就基因型和环境对青钱柳黄酮含量变化的影响以及青钱柳叶片中三萜成分的组织化学定位进行了研究[5-6] ,但不同种源青钱柳叶片中黄酮、三萜和多糖类成分的分布是否存在差异尚不清楚.     

The relationship between population structure and aluminum tolerance in cultivated sorghum

Background

Acid soils comprise up to 50% of the world''s arable lands and in these areas aluminum (Al) toxicity impairs root growth, strongly limiting crop yield. Food security is thereby compromised in many developing countries located in tropical and subtropical regions worldwide. In sorghum, SbMATE, an Al-activated citrate transporter, underlies the Alt_SB locus on chromosome 3 and confers Al tolerance via Al-activated root citrate release.

Methodology

Population structure was studied in 254 sorghum accessions representative of the diversity present in cultivated sorghums. Al tolerance was assessed as the degree of root growth inhibition in nutrient solution containing Al. A genetic analysis based on markers flanking Alt_SB and SbMATE expression was undertaken to assess a possible role for Alt_SB in Al tolerant accessions. In addition, the mode of gene action was estimated concerning the Al tolerance trait. Comparisons between models that include population structure were applied to assess the importance of each subpopulation to Al tolerance.

Conclusion/Significance

Six subpopulations were revealed featuring specific racial and geographic origins. Al tolerance was found to be rather rare and present primarily in guinea and to lesser extent in caudatum subpopulations. Alt_SB was found to play a role in Al tolerance in most of the Al tolerant accessions. A striking variation was observed in the mode of gene action for the Al tolerance trait, which ranged from almost complete recessivity to near complete dominance, with a higher frequency of partially recessive sources of Al tolerance. A possible interpretation of our results concerning the origin and evolution of Al tolerance in cultivated sorghum is discussed. This study demonstrates the importance of deeply exploring the crop diversity reservoir both for a comprehensive view of the dynamics underlying the distribution and function of Al tolerance genes and to design efficient molecular breeding strategies aimed at enhancing Al tolerance.     

沉香叶解剖结构的研究

通过石蜡切片法,光学显微镜观察,研究了沉香叶的解剖结构。结果表明,沉香叶为典型的异面叶,但具有许多旱生特征。表皮由一层排列紧密的形状不规则的表皮细胞组成,细胞外壁角质膜较厚,上表皮角质膜较下表皮的厚3.48μm,下表皮上零星分布着单细胞表皮毛,气孔类型为无规则型,仅分布在下表皮上,微下陷;叶肉组织发达,其间分布着较多的长方晶体,其长轴与表皮垂直;栅栏组织由1～2层圆柱形细胞组成,其外层细胞转化为异细胞,栅栏组织∶海面组织为1∶3.5,下表皮内具有1～2层由异细胞组成的下皮层;主脉发达,有异细胞组成的维管束鞘,具内生韧皮部;叶内具有发达的木质部外纤维。以上特征反映出植物结构与环境的统一性。     

桂花叶解剖结构的研究

通过石蜡切片法制片,光学显微镜观察,研究了桂花叶的形态解剖结构。结果表明,桂花叶为典型的异面叶,表皮由1层排列紧密的形状不规则的表皮细胞构成,细胞外壁角质膜较厚,气孔只分布于下表皮。表皮上被有腺毛,腺毛及其周围排列整齐的表皮细胞形成环式结构。叶肉组织发达,栅栏组织由2～4层排列整齐的圆柱形细胞构成。叶肉中分布有少分枝或不分枝的石细胞,其细胞长轴与表皮垂直。叶脉结构较为复杂,主脉发达。上述特征与复杂多变的环境条件相适应。     

The fine structure and photosynthetic cost of structural leaf variegation

The leaves of some plants display an optical patchiness on their upper side, displaying light- and dark-green areas with high and low reflectance, respectively. In this investigation, we studied the fine structure of the corresponding sectors and we asked whether the lost reflected light entails a photosynthetic cost to these leaves. Four species, i.e. Arum italicum, Ranunculus ficaria, Cyclamen hederifolium and Cyclamen persicum were investigated. Scanning electron microscope examination revealed that epidermal cells of light-green sectors of all species are more bulgy than corresponding cells of neighboring dark-green leaf sectors. The comparative anatomical study revealed that (i) epidermis thickness of the light-green areas and the number of mesophyll cell layers does not differ from those of the adjacent dark-green leaf sectors and (ii) palisade cells of light-green sectors are slightly larger and more loosely arranged, allowing a much higher percentage of intercellular air spaces. The latter histological feature seems to provide the structural basis for the different optical properties between the two leaf sectors. Contrary to expectations, net photosynthetic rates (expressed on a leaf area basis) were similar in the light-green and the dark-green areas of the two cyclamen species. Yet, in C. persicum net photosynthesis was higher in the light-green areas, if expressed on a dry mass basis. The small size of the light-green spots in the rest of the test plants precluded CO₂ assimilation measurements, yet maximum linear photosynthetic electron transport rates displayed no differences between the two sectors in all plants. Thus, the assumption of a photosynthetic cost in the light-green areas was not confirmed. On the contrary, a higher construction cost was evident in the dark-green areas of three species, displaying a significantly higher specific leaf mass, without any photosynthetic benefit. The results on net photosynthesis were compatible with leaf optical properties and pigment levels. Thus, in spite of the considerably higher reflectance of the light-green areas and their lower (yet normal for a green leaf) chlorophyll levels, corresponding differences in absorptance were slight. In addition, dry mass-based pigment contents in dark-green areas were higher, while chlorophyll a/b (in two species) and carotenoid/chlorophyll ratios (in three species) were lower, pointing to a shade adaptation in these sectors. We conclude that in variegated leaves of this kind, dark-green areas are more costly to build and probably less photosynthetically active. We argue that the high pigment contents of dark-green areas establish steep light gradients in the corresponding mesophyll, rendering deeper chloroplast layers more shade adapted.     

不同水分处理下喀斯特土层厚度异质性对两种草本叶片解剖结构和光合特性的影响

为了探究不同水分处理下草本植物对喀斯特土层厚度变化的叶片形态建成和光合生理响应,以黑麦草(Lolium perenne L.)和苇状羊茅(Festuca arundinacea Schreb.)为研究对象,通过盆栽水分受控试验,研究了3种水分处理[正常供水(W_(ck)),减水1组(D1)和减水2组(D2)]下3种土层厚度[浅土组(S_S)、对照组(S_(CK))和深土组(S_D)]对两种草本叶片解剖结构和光合特性的影响。结果表明:(1)正常供水下(W_(ck)),黑麦草和苇状羊茅在浅土组(S_S)的气孔密度和气孔限制值(Ls)均显著高于对照组(S_(CK)),净光合速率(Pn)、胞间CO_2浓度(Ci)和蒸腾速率(Tr)降低;在深土组(S_D),两种植物的气孔密度都有所下降,黑麦草的叶脉密度、Pn和Tr均低于对照组,而苇状羊茅的叶脉密度和Pn表现出增加;(2)D1水分条件下,黑麦草在浅土组的气孔密度较对照组增加,叶脉密度、Pn和Tr均降低,而苇状羊茅的气孔密度有所降低,叶脉密度、Pn和Tr未受到显著影响;在深土组中,黑麦草的气孔密度不变,叶脉密度增加,而Pn和Tr均降低;苇状羊茅的气孔密度降低,但叶脉密度、Pn和Tr均升高;(3)D2水分条件下,两种植物在浅土组的叶脉密度较对照组均增加,气孔密度、Pn和Tr均受到抑制;在深土组,黑麦草的远轴面气孔密度较对照组下降,两种植物的其他指标未受到明显影响。可见,在不同水分条件下,植物的叶片解剖结构和光合特性对不同土层厚度的响应不一,且不同物种间也有差异。总体上随着水分减少,土层厚度降低对植物的光合抑制作用增强,而厚度增加对深根植物的光合促进作用和对浅根植物的光合抑制作用先增强后减弱。植物气孔和叶脉性状特征随水分条件的变化在一定程度上与叶面积和叶片宽度的变化有关。     

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.     

异质性光对野牛草叶片解剖结构的影响

自然界中植物生长所需资源通常呈异质性分布, 具有发达匍匐茎的野牛草(Buchloe dactyloides)在蔓延过程中相连克隆分株常生活在异质性的光环境中。有研究证明, 在异质性光条件下, 植株幼叶的叶片解剖结构受成熟叶所处光照条件的影响, 而异质性光条件下克隆分株的叶片形态解剖结构是否也受相连分株所处光照条件的影响则未见报道。通过设置高光(全光照)和低光(遮阴)两个水平, 对由匍匐茎相连的野牛草克隆分株施以同质和异质性光处理, 研究了异质性光对野牛草叶片解剖结构的影响。结果发现: 在异质性光环境中, 遮阴的野牛草克隆分株的主脉直径、维管束鞘细胞个数、叶片厚度以及近轴侧和远轴侧叶肉细胞的厚度均显著降低; 同质性的低光处理对这些指标则没有显著影响。在异质性光处理下, 未遮阴姊株近轴侧和远轴侧叶肉细胞的厚度以及远轴侧的气孔大小显著增加, 而未遮阴的妹株近轴侧和远轴侧叶肉细胞的厚度、气孔密度和气孔大小、叶片厚度和维管束鞘细胞个数则会降低。同质高光处理下克隆分株近轴侧和远轴侧的气孔密度和气孔大小显著高于同质低光。野牛草克隆分株近轴侧和远轴侧叶肉细胞的厚度、气孔密度和气孔大小受相连分株所处光照条件的显著影响。该研究结果表明: 未遮阴的姊株因为与遮阴的妹株相连而显著受益, 而未遮阴的妹株则因为与遮阴的姊株相连而损耗严重; 在异质性光处理下, 遮阴分株叶片形态上缩减的可塑性生长是为减少维持其存活的消耗, 提高遮阴分株存活率的一种适应性表现。     

Submergence-induced morphological, anatomical, and biochemical responses in a terrestrial species affect gas diffusion resistance and photosynthetic performance

Gas exchange between the plant and the environment is severely hampered when plants are submerged, leading to oxygen and energy deficits. A straightforward way to reduce these shortages of oxygen and carbohydrates would be continued photosynthesis under water, but this possibility has received only little attention. Here, we combine several techniques to investigate the consequences of anatomical and biochemical responses of the terrestrial species Rumex palustris to submergence for different aspects of photosynthesis under water. The orientation of the chloroplasts in submergence-acclimated leaves was toward the epidermis instead of the intercellular spaces, indicating that underwater CO(2) diffuses through the cuticle and epidermis. Interestingly, both the cuticle thickness and the epidermal cell wall thickness were significantly reduced upon submergence, suggesting a considerable decrease in diffusion resistance. This decrease in diffusion resistance greatly facilitated underwater photosynthesis, as indicated by higher underwater photosynthesis rates in submergence-acclimated leaves at all CO(2) concentrations investigated. The increased availability of internal CO(2) in these "aquatic" leaves reduced photorespiration, and furthermore reduced excitation pressure of the electron transport system and, thus, the risk of photodamage. Acclimation to submergence also altered photosynthesis biochemistry as reduced Rubisco contents were observed in aquatic leaves, indicating a lower carboxylation capacity. Electron transport capacity was also reduced in these leaves but not as strongly as the reduction in Rubisco, indicating a substantial increase of the ratio between electron transport and carboxylation capacity upon submergence. This novel finding suggests that this ratio may be less conservative than previously thought.