Gasometric method of water deficit measurement in leaves

A gasometric method was developed for measuring water deficit in leaves. For a leaf at full turgor the amount of water penetrating into the tissue after removing the air from intercellular spaces by means of a vacuum pump, is equal in volume to the gas removed from the intercellular spaces. In a leaf with a water deficit the amount of the infiltrating water is greater than the removed gas volume by the amount egual to the water deficit. Determination of the volumes of the gas removed and penetrating water enables water deficit, if any, to be calculated. Comparative measurements carried out on five plant species confirmed the correctness of the method suggested. Reduction of the measuring time allowed to eliminate completely the sources of errors associated with the growth of tissue and loss of dry weight during respiration.     

Diurnal changes in water deficit and recovery patterns of intact rice leaves under natural and controlled environment conditions

In the natural monsoon environment, the leaves of Bala and Jaya cultivars experienced more than 10 per cent LWD for about 8 and 6 1/2 h in the diurnal cycle, respectively. The maximum LWD of around 14 per cent occurred between 14.30 to 15.30 at the time of minimum relative humidity (EH) and maximum air temperature (Ta). On the other hand, under controlled environment conditions, LWD values of both the cultivars remained below 10 per cent in the entire diurnal cycle. There was a close relationship between LWD and Ta (r = 0.90 for Bala and 0.91 for Jaya) as well as RH (r = -0.95 for Bala and -0.85 for Jaya). The results suggested that RWC values obtained on an interval of 30 min were not enough to observe more than the general trend as a complete recovery of stress achieved within 25 min of rewatering the stressed leaves. Calculations indicated that the determination of LWD with the techniques using excised leaf are likely to incur error as rice leaf may loose water at a rate of 0.3 MPa min⁻¹.     

A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves

Transpiration of cuticular membranes isolated from the lower stomatous surface of Hedera helix (ivy) leaves was measured using a novel approach which allowed a distinction to be made between gas phase diffusion (through stomatal pores) and solid phase diffusion (transport through the polymer matrix membrane and cuticular waxes) of water molecules. This approach is based on the principle that the diffusivity of water vapour in the gas phase can be manipulated by using different gases (helium, nitrogen, or carbon dioxide) while diffusivity of water in the solid phase is not affected. This approach allowed the flow of water across stomatal pores ('stomatal transpiration') to be calculated separately from the flow across the cuticle (cuticular transpiration) on the stomatous leaf surface. As expected, water flux across the cuticle isolated from the astomatous leaf surface was not affected by the gas composition since there are no gas-filled pores. Resistance to flux of water through the solid cuticle on the stomatous leaf surface was about 11 times lower than cuticular resistance on the astomatous leaf surface, indicating pronounced differences in barrier properties between cuticles isolated from both leaf surfaces. In order to check whether this difference in resistance was due to different barrier properties of cuticular waxes on both leaf sides, mobility of 14C-labelled 2,4-dichlorophenoxy-butyric acid 14C-2,4-DB) in reconstituted cuticular wax isolated from both leaf surfaces was measured separately. However, mobility of 14C-2,4-DB in reconstituted wax isolated from the lower leaf surface was 2.6 times lower compared with the upper leaf side. The significantly higher permeability of the ivy cuticle on the lower stomatous leaf surface compared with the astomatous surface might result from lateral heterogeneity in permeability of the cuticle covering normal epidermal cells compared with the cuticle covering the stomatal cell surface.     

NAD kinase activity in wheat leaves under water deficit

Total NAD kinase activity remained unaltered in the drought non-adapted wheat leaves under water deficit, but gradually decreased with water deficit in the adapted ones. The share of the calmodulin-dependent enzyme was significantly higher in the drought-hardened than in non-hardened plants; however, under severe water deficit the activity of the enzyme dropped by half. It seems, therefore, that NAD kinase activity does not limit phosphorylation of NAD in dehydrated plant tissues.     

A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves

In order to examine the transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves, a micro-fluorescence imaging system was devised using a microscope, a CCD camera with an image intensifier, an Ar and a He-Ne laser light source, an image processor, and a microcomputer. A laser light was projected vertically on to the surface of a rice leaf segment at a cut-edge, and scattered light and induced fluorescence were observed at the cut-section from a 90° angle to the axis of the laser beam. The intensity of scattered light showed a maximum at several micrometres depth from the leaf surface and a steep gradient afterwards. Fluorescence reached a maximum crossing with the decline curve of the scattered light. The maximum of fluorescence measured at 741 nm was observed at a greater depth from the leaf surface than that at 687 nm, suggesting that part of the fluorescence of the longer wavelength was emitted due to absorption of fluorescence of the shorter wavelength. Profiles of the scattered light and the chlorophyll fluorescence depended on leaf anatomy.     

Release and aggregation of the light-harvesting complex in intact leaves subjected to strong CO2 deficit

Tobacco plants were subjected to long-term CO₂ deficit. The stress caused photoinhibition of Photosystem (PS) II photochemistry and the aggregation of the light-harvesting complex of PS II (LHC II). The aggregation was shown by the appearance of the characteristic band at 698–700 nm (F₆₉₉) in 77 K fluorescence emission spectra. LHC II aggregates are considered to quench fluorescence and, therefore, the fluorescence yield was determined to verify their quenching capability. PS II photochemistry, measured as F_V/F_M, was largely depressed during first 4 days of the stress. Unexpectedly, the total fluorescence yield increased in this period. Fitting of emission spectra by Gaussian components approximating emission bands of LHC II, PS II core, PS I and F₆₉₉ revealed that mainly the bands at 680 and 699 nm, representing emission of LHC II aggregates, were responsible for the increase of the fluorescence yield. This shows an interruption of the excitation energy transfer between LHC II and both photosystems and, thus, a physical disconnection of LHC II from photosystems. PS II and PS I emissions were not quenched in this period. Therefore, it was concluded that these LHC II aggregates were accumulated out of PS II antenna, and, thus they cannot be involved in dumping of excess excitation. The total fluorescence yield turned to decrease only after the large depression of PS II photochemistry, when LHC II aggregation was considerably speeded up and the fluorescence yields of PS I and II turned to decline.     

结实期土壤水分亏缺影响水稻籽粒灌浆的生理原因

通过分析结实期土壤水分亏缺对水稻(Oryza sativa)籽粒中蔗糖向淀粉合成的生理代谢中关键酶活性及籽粒灌浆的调节作用, 探讨土壤水分亏缺影响水稻籽粒灌浆的生理机制。结果表明, 适度土壤水分亏缺诱导了灌浆高峰期(花后15-20天)水稻籽粒中蔗糖合成酶、腺苷二磷酶葡萄糖焦磷酸化酶、可溶性淀粉合成酶及淀粉分支酶活性的增加, 提高了籽粒灌浆中前期(花后10-20天)籽粒中淀粉积累速率和籽粒灌浆速率。但在灌浆后期(花后20-30天)籽粒中, 上述关键酶活性下降较快, 籽粒活跃灌浆期明显缩短, 灌浆前中期灌浆速率的增加不能完全补偿灌浆期缩短带来的同化物积累损失, 导致水分亏缺处理水稻籽粒充实不良, 结实率、籽粒重和产量显著降低。研究认为, 灌浆期土壤水分亏缺引起的灌浆后期籽粒中蔗糖向淀粉合成代谢中一些关键酶活性快速下降和籽粒内容物的供应不足是籽粒淀粉积累总量减少、粒重降低的主要生理原因。     

Deciphering genetic variations of proteome responses to water deficit in maize leaves

The proteome of the basal part of growing Zea mays leaves was analyzed from 4 to 14 d after stopping watering and in well watered controls. The relative quantity of 46 proteins was found to increase in leaves of plants submitted to water deficit. Different types of responses were observed, some proteins showing a constant increase during water deficit, while others showed stabilization after a first increase or a transient increase. Isoforms encoded by the same gene showed different responses. The response to water deficit showed genetic variation. Some increased proteins were induced specifically in one of the two studied genotypes (e.g. ASR1) while others were significantly induced in both genotypes but to a different level or with different kinetics. Analyses of relations between protein quantities, relative water content (RWC) and abscisic acid (ABA) concentration allowed us to show that the quantitative variation of some proteins (e.g. ABA45 and OSR40 proteins) was linked to differences in ABA accumulation between the genotypes. Other proteins showed genetic variations that were not related to differences in water status or ABA concentration (e.g. a cystatin). Data obtained from these experiments, together with data from other experiments, contribute to the characterization of maize proteome response to drought in different conditions and in different genotypes. This characterization allows the search for candidate proteins, i.e. for protein whose genetic variation of expression could be partly responsible for the variability of plant responses to drought.     

A technique for measurement of retrograde coronary blood flow in intact anesthetized dogs.

A technique for measurement of retrograde coronary blood flow in intact anesthetized dogs is described. Occlusion of the coronary artery is produced by the inflation of a small rubber balloon at the tip of a no. 9 cardiac catheter placed under fluorescopy in a branch of the left coronary artery. Blood which bleeds back from the occluded coronary artery through the no. 9 catheter is diverted into a small reservoir of 1-ml capacity. The time to fill this reservoir is recorded electrically. Retrograde coronary blood flow is calculated from the time required to fill this reservoir. Results indicate good repeatability of meadurements. The technique seems to be a simple, adequate, and convenient means for assessing agents for possible vasodilator action on the collateral circulation in intact animals.     

Proteomics to identify pathogenesis-related proteins in rice roots under water deficit

Upland and lowland rice (Oryza sativa L.) showed different mechanisms of water stress resistance. Hydroponically grown 3-week-old seedlings of a lowland variety IR64 and an upland variety were exposed to 15% polyethylene glycol (PEG-6000). After 7 d of treatment, IR64 maintained high relative water content and developed a well-branched root. Therefore, IR64 had better water-deficit tolerance than Azucena under PEG treatment. To identify water-deficit-responsive proteins associated with the tolerance differences between two ecotypes, a comparative proteomic analysis of roots was conducted. Out of 700 proteins reproducibly detected on two-dimensional electrophoresis gels, 65 proteins exhibited significant changes in at least one ecotype at 48 h of water deficit. Only 15 proteins showed different responses to water deficit between the two ecotypes. Twelve proteins were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry, which involved in energy and metabolism, protein processing and degradation, detoxification and pathogenrelated (PR) proteins, i.e. PR-1a, RSOsPR10 and JIOsPR10. All three PR proteins were induced more strongly in IR64 than in Azucena by water deficit at both protein and mRNA level. The results suggested that PR-1a, RSOsPR10 and JIOsPR10 may play important roles in protecting root cells against water deficit in rice.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Respiratory energy demand for protein turnover and ion transport in wheat leaves upon water deficit

The effect of water stress on the respiratory energy demand for the main biosynthetic and transport processes was estimated in the leaves of spring wheat ( Triticum aestivum L. cv. San Pastore) acclimated and non-acclimated to drought. ATP-consuming processes were assessed from the effects of selective inhibitors of RNA synthesis, protein synthesis and proteolysis, Ca²⁺-ATPase and P-type ATPases on respiration. The proportions of energy consumed by these processes were compared with the theoretical ATP production calculated from the rate of oxygen consumption measured manometrically. Respiratory energy production increased significantly in both acclimated leaves and in leaves stressed by drought. In the fully grown wheat leaves, Ca²⁺-dependent reactions and protein turnover consumed about 37% and 34% of the total respiratory energy, respectively. The costs of ion transport constituted another 15% of the total ATP production. Both acclimation and drought stress in non-acclimated leaves resulted in a decrease of leaf sensitivity towards inhibitors of RNA and protein syntheses as well as a decrease in Ca²⁺-mediated processes; but also in an increase of leaf sensitivity towards inhibitors of proteolysis and ouabain-sensitive ATPase in non-acclimated plants. This indicates a shift in ATP input into the energy-requiring processes towards greater expenses for ion transport upon water deficit. However, in acclimated leaves under drought stress, distribution of respiratory energy became almost the same as in control plants.     

A photographic technique for investigating the splashing of water drops on leaves

The formation of splash from water drops falling onto bean leaves was investigated by multiple exposure flash photography. A relatively simple technique was developed which recorded up to four images at 2 ms intervals, of a splash on one photograph. The images were recorded on colour reversal film, and the flash sequence was colour coded to aid the interpretion of the superimposed images. Analysis of the photographs allowed measurement of the initial speed and angle of ejection of different sized splash droplets. Droplets were ejected at speeds of up to about 10 m s^-1although most speeds were less than about 3 m s^-1, and small droplets tended to travel faster than large ones. Droplets were ejected at all angles but the majority were ejected almost horizontally; there was little correlation between droplet size and angle of ejection. Deposition patterns estimated from calculated droplet trajectories compared well with measured ones.     

比重法定量局部脑组织含水量方法的改良

目的：在保证比重法测量脑组织含水量准确性的基础上,改良其测量方法。方法：观察使用密度计建立的液体比重柱的稳定性和梯度均匀性;比较低温脑组织采集法与冷冻法对比重法测定脑组织含水量的影响。结果：采用密度计制备的液体比重柱的稳定性和梯度均匀性提高;低温脑组织直接采集法减少比重法测量脑组织含水量的系统误差。结论：采用密度计和低温直接标本采集法不仅提高比重法测定脑组织含水量的准确性,且简化实验步骤。     

Photochemical heat-shock response in common bean leaves as affected by previous water deficit

The heat sensitivity of photochemical processes was evaluated in the common bean (Phaseolus vulgaris) cultivars A222, A320, and Carioca grown under well-watered conditions during the entire plant cycle (control treatment) or subjected to a temporal moderate water deficit at the preflowering stage (PWD). The responses of chlorophyll fluorescence to temperature were evaluated in leaf discs excised from control and PWD plants seven days after the complete recovery of plant shoot hydration. Heat treatment was done in the dark (5 min) at the ambient CO₂ concentration. Chlorophyll fluorescence was assessed under both dark and light conditions at 25, 35, and 45°C. In the dark, a decline of the potential quantum efficiency of photosystem II (PSII) and an increase in minimum chlorophyll fluorescence were observed in all genotypes at 45°C, but these responses were affected by PWD. In the light, the apparent electron transport rate and the effective quantum efficiency of PSII were reduced by heat stress (45°C), but no change due to PWD was demonstrated. Interestingly, only the A222 cultivar subjected to PWD showed a significant increase in nonphotochemical fluorescence quenching at 45°C. The common bean cultivars had different photochemical sensitivities to heat stress altered by a previous water deficit period. Increased thermal tolerance due to PWD was genotype-dependent and associated with an increase in potential quantum efficiency of PSII at high temperature. Under such conditions, the genotype responsive to PWD treatment enhanced its protective capacity against excessive light energy via increased nonphotochemical quenching.     

Upland rice and lowland rice exhibited different P/P expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations.To further understand the aquaporin function in plants underwater stress,the expression of a subgroup of aquaporins,plasma membrane intrinsic proteins(PIPs),was studied at boththe protein and mRNA level in upland rice(Oryza sativa L.cv.Zhonghan 3)and lowland rice(Oryza sativa L.cv.Xiushui63)when they were water stressed by treatment with 20% polyethylene glycol(PEG).Plants responded differently to20% PEG treatment.Leaf water content of upland rice leaves was reduced rapidly.PIP protein level increased markedlyin roots of both types,but only in leaves of upland rice after 10h of PEG treatment.At the mRNA level,OsPIP1;2,Os-PIP1;3,OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1;2 and OsPIP1;3 in leaves were significantly up-regulatedin upland rice,whereas the corresponding genes remained unchanged or down-regulated in lowland rice.Meanwhile,weobserved a significant increase in the endogenous abscisic acid(ABA)level in upland rice but not in lowland rice underwater deficit.Treatment with 60μM ABA enhanced the expression of OsPIP1;2,OsPIP2;5 and OsPIP2;6 in roots andOsPIP1;2,OsPIP2;4 and OsPIP2;6 in leaves of upland rice.The responsiveness of PIP genes to water stress and ABAwere different,implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signalingpathways during water deficit.     

Effects of plant water deficit on the daily carbon balance of leaves of cacao seedlings

The daily carbon balance of individual source leaves of Theobroma cacao L. seedlings was measured at 2- to 3-day intervals during a 19-day period of increasing plant water deficit and during an 8-day period of recovery following rewatering. In each case, responses of stressed seedlings were compared to those of irrigated controls. Leaves of irrigated cacao seedlings assimilated approximately 41 mg carbohydrate dm^-2 during 12-h photoperiods, and exported an average of 34 mg carbohydrate dm^-2 during 24-h measurement cycles. The rate of carbon export from cacao leaves was sharply reduced as leaf water potential (ψ) declined between -0.8 and -2.0 MPa. Further, the rate of export was closely associated with the net assimilation rate (A), with export capacity being severely reduced as A fell to near zero. Net accumulation of dry matter occurred as long as A remained greater than approximately 20 mg carbohydrate dm^-2 over the 12-h photoperiod, but at lower assimilation rates, export exceeded concomitant assimilation. Carbon export continued at the expense of leaf carbon reserves as photoassimilation fell to near zero during periods of severe water stress (ψ < -2.0 MPa). Night respiration rate was independent of plant water status.