Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

Structure and function of the elongation sink in the stems of higher plants I. Effects of anoxia and IAA on the growth rate and the spatially separate electrogenic ion pumps

Two spatially separate membrane potentials of the parenchymasymplast (7, 8) and the rate of elongation growth were measuredsimultaneously in bean hypocotyl segments. Both the membrane potentials, V_ps (electric potential differencebetween parenchyma symplast and organ surface) and V_px (electricpotential difference between parenchyma symplast and xylem),were rapidly depolarized by anoxia, and were repolarized withre-aeration. Anoxia reduced the growth rate by about 85%, andre-aeration restored it. Changes in the membrane potentialspreceded those in the growth rate by 30–50 sec. About 8 min after the application of aerosol generated from10^–3 M indole acetic acid (IAA) solution to the segments,V_ps began to hyperpolarize, and the growth rate increased witha delay of several minutes after the potential change and subsequentlybecame ten times the control rate. This hyperpolarization ofV_ps was due to the increase in the activity of the respiration-dependentelectrogenic ion pump at the outer surface membrane of the parenchymasymplast. A clear correlation was observed between the growthrate and pump activity (V_ps) with the change in IAA concentration. (Received December 6, 1979; )     

Water Transport during Acid-Induced Growth of Excised Hypocotyl Sections of Vigna unguiculata under Xylem Perfusion

Elongation growth of abraded hypocotyl sections of Vigna unguiculataunder xylem perfusion was markedly promoted a few minutes afterthe application of an acid aerosol generated from a solutionof HCl. At the beginning of the acid-induced growth, intracellularpressure (Pⁱ) began to decrease and the membrane potential betweenthe symplast and the xylem apoplast (V^px) began to depolarize.Subsequently, Pⁱ and V^px remained at a reduced level and a depolarizedlevel, respectively, while the promotion of elongation growthcontinued for more than 4 hours. The electrogenic componentof the xylem membrane potential (V^px_act) gradually increasedto about twice that before acid treatment. There was a closecorrelation between the enhanced growth and the decrease inintracellular pressure within 30 min after application of acidbut little correltion after 60 min. By contrast, there was littlecorrelation between the promotion of growth and the activityof the xylem pump after 30 min while a close correlation wasobserved after 60 min. It is inferred that the acid-induced activation of water uptakeconsists of two major processes, in series, that are drivenby different forces: the rapid uptake of water for more than30 min, driven by hydrostatic force generated by loosening ofcell walls; and a long-lasting enhancement of water uptake forat least 4 h, which is driven by osmotic force that is generatedby the canal system within the xylem. (Received October 17, 1994; Accepted January 23, 1995)     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis VI. The dual structure of radial electrogenic activity

The electrophysiological structure in bean hypocotyl was investigatedby the intracellular electrode method in combination with surfaceelectric potential (V_s) measurement and respiratory inhibitionby anoxia, with special reference to the membrane transportof ions and the formation of an absorption centre in the elongating(E) zone. The radial potential difference (V_sx: electric potentialdifference between the organ surface and a xylem vessel), onwhich axial distribution of V_a was dependent, comprised twocomponents; V_ax=V_px–V_ps. |V_px| (the potential differencebetween the inside of a parenchyma symplast and a xylem vessel)was at a maximum in the E-zone, while |V_ps| (the intracellularelectric potential with respect to the organ surface) was largestin the G-zone (mature zone), resulting in the characteristicdistribution pattern of V_s with a minimum in the E-zone. Therewere two independent electromotive forces which were both partiallydependent on respiration; one corresponding to V_ps located atthe surface of the parenchyma symplast (P) and the other toV_px located between P and the xylem (X). The electrogenic componentof Vpx was relatively small both in the hook (H) zone and theG-zone, but maximal in the E-zone of the hypocotyl. This resultwas consistent with the emergence of a maximum pH differencebetween P and X in the E-zone, where accumulation of K⁺ andwater were at a maximum, suggesting maximum activity of an H⁺-pumpextruding protons from P into X in exchange for K⁺. (Received July 17, 1978; )     

Regulation of Elongation Growth of Excised segments of Vigna Hypocotyl under Osmotic Stress in the Absence or Presence of Absorbable Solute

The regulation of elongation growth in excised segments of Vignahypocotyl under osmotic stress was investigated by means ofthe xylem perfusion and pressure-jump method. When subjectedto osmotic stress in the absence of absorbable solutes (100mM sorbitol only) or in the presence of absorbable solutes (70mM sorbitol plus 30 mM sucrose, or 70 mM sorbitol plus 15 mMKC1), the hypocotyl segments immediately began to shrink. Thehyper-polarizations of the transmembrane potentials (V_px andV_ps) took place at once. Within 40–60 minutes, the segmentsresumed growth. In the presence of absorbable solute, therewas an obvious increase in the effective turgor (Pⁱ–Y'),but the physiological wall extensibility () increased only slightly.Conversely, in the absence of absorbable solute, increasedsignificantly but (Pⁱ–Y') decreased. The results suggestthat the recovery of growth of an excised segment under osmoticstress is mainly due to the change in in the absence of absorbablesolute, and to the change in (P¹–Y') in the presence ofabsorbable solute, and that the two respiration-dependent protonpumps play important roles in these recovery processes. (Received April 28, 1989; Accepted August 24, 1989)     

Effects of Osmotic Stress, Salt Stress and IAA on the Regeneration of the Resting Membrane Potential after Excision of a Segment from an Intact Plant

Effects of osmotic stress, salt stress and IAA on the regenerationprocess of the transmembrane potential across the xylem/symplastinterface (V_px) of newly excised hypocotyl segments of Vignaseedlings were examined by means of the xylem perfusion method.It took about 8 h under ordinary conditions for an excised segmentto regenerate a membrane potential comparable with that of anintact seedling. Osmotic stress imposed by perfusion of 100-200mM sorbitol solution seemed to accelerate this process. Thiseffect diminished with the removal of sorbitol from the perfusionsolution. The increase in the negativity of V_px in this regenerationprocess resulted from the increases in both passive (V_px inN₂) and respiration-dependent components (V_px). NaCl (50–100mM) did not accelerate the regeneration of the total membranepotential, but significantly promoted an increase in V_px, i.e.electrogenic xylem pump activity. Perfusion of KC1 (50–100mM) or IAA (10^–4M) shortened the regeneration phase upto 2–3 h. The increase in total V_px under salt stressor IAA mainly resulted from the increase of V_px. The effectof those agents on the V_px is discussed, as is the questionof whether the increase in PD after excision should be interpretedas a recovery or regeneration from injury by excision, or asa so-called release from hormonal control. (Received September 3, 1987; Accepted March 14, 1988)     

Adaptive Growth Responses to Osmotic Stress of Hypocotyl Sections of Vigna unguiculata: Roles of the Xylem Proton Pump and IAA

The growth responses to osmotic stress of hypocotyl sectionsof Vigna unguiculata were studied by the xylem perfusion method.Hypocotyl sections shrank upon exposure to osmotic stress. Sectionsshowed no adaptive responses to osmotic stress when they werein an IAA-depleted condition as a result of perfusion with solutionsthat lacked IAA for 3–4 h. The correlation between thegrowth rate and the membrane potential of the xylem/symplastboundary (V^px) was very limited in the absence of IAA. By contrast,hypocotyl sections showed distinct adaptive responses to osmoticstress after perfusion with solutions that contained 10 µMIAA. In the presence of IAA, V^px increased in the negative directionand growth resumed in spite of the osmotic stress. The growthrate was closely correlated with the xylem membrane potential.Hyper-polarization of the membranes of the xylem/symplast boundaryalways preceded the recovery of growth under osmotic stress.It appears that IAA is essential for the adaptive recovery ofgrowth under osmotic stress and, moreover, that the xylem protonpump plays an indispensable role in modulating the growth ofhypocotyl sections. This result confirms prediction of an earliersimulation study using the apoplast canal model [Katou and Furumoto(1986) Protoplasma 133: 174, Katou and Enomoto (1991) PlantCell Physiol. 32: 343]. (Received June 27, 1996; Accepted October 28, 1996)     

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V _ps andV _px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO₂. Prolonged exposure of the hypocotyl to high concentrations of CO₂ (above 10%) caused depolarization of membrane potentials above the level before CO₂ introduction. When CO₂ was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO₂ as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO₂-induced maximum hyperpolarization ofV _ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO₂ may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.Symbols and abbreviations V _sx electric potential difference between the surface (S) and the xylem (X) of the hypocotyl - V _px electric potential difference between the inside of a parenchyma cell (P) andX - V _ps electric potential difference betweenP andS - V _ps (CO₂, max) the maximum value of CO₂-induced hyperpolarization ofV _ps - GR(CO₂, max) the maximum value of CO₂-induced increase of the growth rate - IAA indole-3-acetic acid     

Effects of Gibberellin on Auxin-Induced Hyperpolarization of Cell Membranes in Hypocotyls of Vigna unguiculata

Auxin activates pumping of protons from the symplast to theapoplast and causes hyperpolarization of the symplast membranein the elongation zone of Vigna stems prior to the accelerationof growth. This auxin-induced hyperpolarization has been studiedin most cases in hypocotyl segments excised from the elongationzone. In the present study, mature-zone segments were perfusedwith IAA by the xylem perfusion technique in an effort to determinewhether or not IAA has any effects in the mature zone. Althoughno hyperpolarization of the symplast membrane was observed uponthe perfusion with auxin alone, auxin-induced hyperpolarizationwas observed when mature-zone segments had been pretreated withGA₃, in the absence of an increase in the growth rate. Theseresults suggest that cells in the mature zone have lost theability to activate the proton-pumping machinery in responseto auxin but that this ability can be restored by treatmentwith GA₃. This effect of GA₃ suggests the possibility that theconcentration of gibberellin in a tissue controls one of thecell's responses to auxin, namely, activation of the protonpump. (Received January 10, 1994; Accepted June 11, 1994)     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis V. Electrogenic activity of the parenchyma cells in hypocotyl segments

Intracellular potential of parenchyma cells (V_ps) in the hypocotylsegment of Vigna sesquipedalis was initially low after excision,then gradually increased to a more negative level. ThereafterV_ps could be remarkably reduced under anoxia then recoveredwith reaeration, accompanied by several cycles of damped oscillations.Both the rapidity of the decrease of V_ps caused by anoxia andits temperature dependency suggest an electrogenic mechanism.No marked spatial differences along the germ axis and the radiusof hypocotyl were observed in the V_ps level and its electrogeniccomponent when the reference electrode was placed on the surfaceof the hypocotyl segment. V_ps also decreased rapidly in an atmospherecontaining 80% CO+20% O₂ in the dark, then was recovered immediatelyin the light or spontaneously but very slowly in the dark. Theextent of the decrease of V_ps caused by CO depended on the growthactivity of the cell and the time lapse after excision. Theseresults suggest the possible conversion or replacement of theterminal oxidase. Sometimes phenomena resembling those of actionpotentials occurred spontaneously or during the reduction ofV_ps due to inhibition of the energy metabolism. V_ps in the elongatingregion varied transiently with the change in illumination. Interrelationshipbetween V_ps and the surface resting potential is discussed. (Received July 20, 1977; )     

Effects of Osmotic Stress, Ionic Stress and IAA on the Cell-Membrane Resistance of Vigna Hypocotyls

The cell-membrane resistance (R_m) of Vigna hypocotyls was examined,and the effects of osmotic stress, ionic stress and IAA on R_mwere investigated. R_m decreased by 64 to 77% under osmotic stressin the presence of absorbable solutes (40 mM sorbitol, 15 mMKC1, 30 mM sucrose; or 40 mM sorbitol, 15 mM KC1, 30 mM sucroseplus 10^–4 M IAA) or under ionic stress (50 mM NaCl or50 mM KC1). R_m was not changed by perfusion with 10^–4M IAA. Therefore, the hyper-polarizations of the membrane potentialobserved in both cases should be ascribed totally to the activationof the electrogenic proton pump. Although R_m showed an increaseof 1.6 fold when the hypocotyls were subjected to osmotic stress(100 mM sorbitol or 100 mM sorbitol plus 10^–4 M IAA),83.6% or 92.4% of the hyperpolarization of the membrane potential(V_px was also the result of the activation of the pump. Theresults, calculated on the basis of the current source model,support the viewpoint that the hyperpolarization of the cellmembrane potential of Vigna hypocotyls under osmotic stress,ionic stress or in the presence of IAA is an expression of theactivation of the proton pump, and is not caused by an increasein R_m. ¹ Present address: Researchers and Planners of Natural Environment, Yotsugi Bldg. (2F), 1-5-4 Horinouchi, Suginami-Ku, Tokyo,166 Japan ² Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2 Seto, Kanazawa-Ku, Yokohama, 236 Japan (Received February 14, 1991; Accepted July 24, 1991)     

Biochemical Properties of the Plasmalemma ATPase of Jerusalem Artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Enzymological properties of the ATPase of plasmalemma-enrichedfractions obtained from the parenchyma of Jerusalem artichoke(Helianthus tuberosus L.) tubers were studied in relation tothe morphogenetic properties of the tubers. Preparations fromdormant tubers showed higher V_max and K_m values than those fromnon-dormant tubers. These differences persisted after solubilizationof the enzyme with octylglucoside, but were slightly modifiedafter addition of K$ to the reaction medium. The implicationsof these results are discussed in relation to the morphogeneticproperties of the tubers (Received August 24, 1987; Accepted March 19, 1988)     

Demonstration of the root surface electrogenic ion pump activity revealed from the seasonal inversion in the phase relation between electro-radicogram and the diurnal oscillation of air temperature in a field tree (Diospyros kaki)

Re-examination of the electro-radicogram (ERG) obtained during past 10 years research (Masaki and Okamoto in Trees (Berl) 21:433–442, 2007) enabled us to discriminate the excess activity of the electrogenic ion pump in the root surface cell membrane over that of the xylem pump during most of the foliate phase. The trans-root electric potential (TRP) is defined as the difference between V _ps (electric potential difference between symplast and bulk water phase surrounding the root) and V _px (electric potential difference between symplast and xylem apoplast). The diurnal oscillation of TRP followed that of the air temperature and/or light intensity with a delay of several hours during defoliate phase. This means the superiority of the electrogenic activity of the xylem pump over that of the root surface pump. However, after leaf expansion, TRP began to oscillate inversely with the temperature change with a short delay, indicating the superiority of the electrogenic activity of the surface ion pump over that of the xylem pump. An experimental lumbering of the surroundings of the kaki tree in foliate phase prominently increased the ERG amplitude, keeping the inverted phase relation, with the increase in transpiration caused by the increased illumination. An incidental sudden fall of the temperature and illumination caused an inverse reaction.     

The Cellular Pathway of Short-distance Transfer of Photosynthates and Potassium in the Elongating Stem of Phaseolus vulgaris L. A Structural Assessment

The potential cellular pathway of radial transfer of photosynthateand potassium delivered in the phloem to the elongation zone(apical 0.5–2.5 cm) of internode 2 ofPhaseolus vulgarisL. seedlings was elucidated. This was achieved using ultrastructuralobservations of the cell types that constitute the radial pathwayand estimates of potential sucrose and potassium fluxes throughthe cross-sectional area of interconnecting plasmodesmata andacross the plasma membrane surface areas of selected cell types.The investigation relied on predicting the relative roles ofthe mature and developing sieve elements as conduits for theaxial delivery of solutes to the elongation zone. In turn, thesepredictions led to formulation of two transport models whichwere subsequently evaluated. It was found that unloading ofsucrose and potassium from the protophloem sieve elements cannotbe through the symplast due to the absence of plasmodesmata.On the other hand, mature metaphloem sieve element-companioncell complexes have the potential capacity to unload eitherthrough the stem symplast or apoplast. The potential symplasticroute is proposed to be via the companion cells to the adjacentlarge phloem parenchyma cells. Continued radial transfer couldoccur either by exchange to the stem apoplast from the largephloem parenchyma cells or continue in the symplast to the groundtissues. It was further predicted that sucrose utilized forthe development of the procambial/small phloem parenchyma cellscould be delivered axially by them and not by the mature sieveelements. Phaseolus vulgaris ; apoplast; elongating stem; photosynthates; potassium; transport; symplast     

Biophysical characterization of zebrafish connexin35 hemichannels

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels (I_hc) that were activated and increased by stepwise depolarization of membrane potential (V_m) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (_hc: 250–320 pS). The _hc of Cx35 hemichannels exhibited a pronounced V_m dependence; i.e., _hc increased/decreased with relative hyperpolarization/depolarization (_hc was 72 pS at V_m = –100 mV and 35 pS at V_m = 100 mV). Extrapolation to V_m = 0 mV predicted a _hc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of 24 pS. Channel gating was also V_m dependent: open time declined with negative V_m and increased with positive V_m. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo. connexin; gating; retina     

Effects of anoxia and high CO2 concentration on the electrogenic activity of leaf cell membrane in the dark

Resting membrane potential of a single cell in Antirrhinum leafdepolarized rapidly by about 40% under anoxia in the dark andrecovered to the normal level with reaeration. V_m in the dark comprised two fractions; one was passive diffusionpotential and the other was intimately dependent on aerobicmetabolism. The rapidity of the depolarization due to anoxiasuggested the possible functioning of an electrogenic mechanism,although the level of V_m was not much higher than the equilibriumpotential of K⁺ across membrane. 50% CO₂ in air brought about slightly more complex responses:a slight transient hyperpolarization and a subsequent depolarization.Sometimes one more transient depolarization took place as a"CO₂-off" effect. The total depolarization span was equal tothat caused by anoxia. 50% CO₂ under anoxia did not evoke furtherdepolarization but only hyperpolarization. (Received July 7, 1976; )     

Hormone Effects on the Membrane Potential and on Sucrose-Induced Depolarization of Young Citrus Leaves

The effect of IAA, GA₃ and ABA on transmembrane potential difference(Em) and on sucrose-induced depolarization has been studiedin young Citrus leaves. The addition of any of these hormonesto the perfusion solution (short-term experiments) did not affectEm or sucrose-induced depolarization. Hormonal treatments ofyoung leaves on the tree resulted, after 4 to 16 days (long-termexperiments), in an increase of Em for GA₃- and ABA-treatedleaves, while in IAA-treated ones no hyperpolarization was found.Only in ABA treated leaves this membrane hyperpolarization couldbe related to an enhancement of sucrose uptake. (Received April 28, 1992; Accepted September 21, 1992)     

Role of K+ and Cl- in Osmotic Adjustment in Roots and Hypocotyls of Intact Mung Bean Seedlings

It was confirmed that osmotic adjustment occurred in young intactmung bean (Vigna mungo (L.) Hepper) seedlings exposed to highosmotic pressure stress. Root growth was not affected by osmoticpressure of less than 200 mOsra in the external solution, althoughhypocotyl growth was conspicuously reduced. Under this moderateosmotic stress, intracellular K⁺ concentration, [K⁺]_i, increaseddramatically during the osmotic adjustment in all the regionsof the root, but the intracellular Cl^– concentration,[Cl^–]_i, increased only in the aged mature region of theroot (28–33 mm from the root tip). About a half of theintracellular osmotic pressure in the aged mature region ofthe root could be ascribed to the contributions of [K⁺]_i and[Cl^–]_i, but in the hypocotyl, [K⁺]_i only contributed slightlyto the osmotic adjustment. (Received June 18, 1986; Accepted August 26, 1986)     

Nutrient-limited growth rates: quantitative benefits of stress responses and some aspects of regulation

Young sunflower plants (Helianthus annuus L.) under stress oflow nitrate or phosphate availability exhibited increases inroot: shoot ratio and in kinetic parameters for uptake. Theyshowed no significant changes in photosynthetic utilizationof either nutrient. Increases in root: shoot ratio were achievedby early and persistent suppression of shoot growth, but notroot growth. Affinity for phosphate uptake, 1/K_m(P), increasedwith phosphate stress, as did affinity for nitrate uptake, 1/K_m(N),with nitrate stress. Maximal uptake rate, V_max, for phosphateuptake increased with phosphorus stress; V_max for nitrate didnot increase with nitrogen stress. Phosphate V_max was relatedstrongly to root nutrient status. Decreases in V_max with plantage were not well explained by changes in age structure of roots.Estimated benefits of acclimatory changes in root: shoot ratioand uptake kinetics ranged up to 2-fold increases in relativegrowth rate, RGR. The relation of RGR to uptake physiology followedpredictions of functional balance moderately well, with somesystematic deviations. Analyses of RGR using growth models implyno significant growth benefit from regulating Vmax, specifically,not from down-regulating it at high nutrient availability. Quantitativebenefits of increases in root: shoot ratio and uptake parametersare predicted to be quite small under common conditions whereinnutrient concentrations are significantly depleted by uptake.The root: shoot response is estimated to confer the smallestbenefit under non-depleting conditions and the largest benefitunder depleting conditions. Even then, the absolute benefitis predicted to be small, possibly excepting the case of heterogeneoussoils. Depleting and non-depleting conditions are addressedwith very different experimental techniques. We note that atheoretical framework is lacking that spans both these cases,other than purely numerical formulations that are not readilyinterpreted. Key words: Nutrient stress, nutrient uptake, nutrient use efficiency, relative growth rate, Helianthus annuus