Helianthus nighttime conductance and transpiration respond to soil water but not nutrient availability

1 We investigated the response of Helianthus sp. nighttime conductance (g night ) and transpiration 2 (E night ) to soil nutrient and water limitations in nine greenhouse studies. The studies primarily 3 used wild Helianthus annuus L. , but also included a commercial and early domesticate of H. 4 annuus, and three additional wild species (H. petiolaris Nutt. , H. deserticola Heiser , and H. 5 anomalus Blake ) . Well watered plants of all species showed substantial g night (0.023-0.225 mol 6 m -2 s -1 ) and E night (0.29-2.46 mmol m -2 s -1 ) measured as instantaneous gas exchange. Based on 7 the potential for transpiration to increase mass flow of mobile nutrients to roots, we hypothesized 8 that g night and E night would increase under limiting soil nutrients, but found no evidence of 9 responses in all six studies testing this. Based on known daytime responses to water limitation, 10 we hypothesized that g night and E night would decrease when soil water availability was limited, 11 and results from all four studies testing this supported our hypothesis. We also established that 12 stomatal conductance at night was on average five times greater than cuticular conductance. 13 Additionally, g night and E night varied nocturnally and across plant reproductive stages while 14 remaining relatively constant as leaves aged. Our results further the ability to predict conditions 15 under which nighttime water loss will be biologically significant and demonstrate that for 16 Helianthus , g night can be regulated.


Introduction 18
It is widely accepted that plants regulate stomatal aperture both to minimize water loss 19 for a given amount of carbon assimilated and to minimize xylem cavitation (Cowan, 1977;20 Sperry, 2000). C 3 and C 4 plants fix carbon during the day and lose water from leaves as an 21 unavoidable cost of getting CO 2 to the site of carboxylation. Although these plants are generally 22 expected to close their stomata at night to conserve water when carbon gain is not occurring, 23 significant nighttime leaf conductance (g night ) and transpiration (E night ) have been observed in 24 many C 3 species across a wide range of habitats (see Musselman and Minnick, 2000;Caird et al., 25 submitted-a for reviews). Reported rates for g night typically range from 0.01 to 0.25 mol m -2 s -1 26 and can represent greater than 50% of daytime conductance (g day ). Nighttime transpiration 27 depends on both g night and leaf-to-air vapor pressure deficit (VPD l ), but is usually 5-15% of 28 daytime transpiration (E day ). To date most studies document the magnitude of g night and E night and 29 several have correlated these traits with environmental or physiological variables (Benyon et  water flux to the rhizoplane minimizes or eliminates the formation of a nitrate depletion zone 37 around plant roots when conditions are appropriate for E night (Barber and Cushman, 1981; 38 Barber, 1995 on long distance nitrogen transport within the xylem, not with mass flow delivery to roots. Thus, 42 increased nutrient acquisition may represent a benefit that counters the cost of water loss at night. 43 If nighttime water loss increases nutrient acquisition then plants may benefit from the 44 ability to regulate g night in response to nutrient conditions. The effects of nitrate availability on 45 g day and E day have been investigated and are variable (Chapin, 1990;Fredeen et al., 1991;Ciompi 46 et al., 1996;Cechin and Fumis, 2004). Potential regulatory pathways are still being debated 47 treatments found that g night declined in response to nutrient additions (Ludwig et al., 2006;Scholz 49 et al., 2007). However, the experimental designs of these studies did not permit direct effects 50 due to reduced plant demand for nutrient acquisition regulating g night to be separated from 51 indirect effects of plant size or water status. More studies are needed that experimentally 52 manipulate soil nutrient availability and test its effect on g night and E night , independent of 53 confounding variation in soil and plant water potential. 54 During the day stomatal conductance is regulated with respect to changing soil water 55 potential and atmospheric demand, to minimize use of available water during CO 2 uptake and 56 maintain soil to leaf hydraulic continuity (Sperry et al., 2002). To further optimize use of limited 57 soil water, regulation may also occur at night, reducing g night  However, in both of these cases direct effects of reproductive stage and leaf age cannot be 72 differentiated from additional variables such as plant size and age. Controlled studies are needed 73 to accurately assess the role of plant reproductive stage and leaf age on g night . 74 Most measures of plant water loss include loss across both the cuticular and stomatal 75 pathways operating in parallel. Because cuticular conductance (g cuticular ) is very small compared 76 to daytime conductance through open stomata (g stomata ), its contribution to g day has traditionally 77 been ignored. However, when considering much lower magnitude g night and E night , cuticular 78 losses may represent a substantial portion of the total measurement. Estimates of g cuticular , 79 Response of g night and E night to soil nutrient and water manipulation 115 Six studies applied a soil nutrient treatment, four of which only manipulated soil nitrate 116 (Table 1). There was no effect of nutrient limitation on g night and E night in any of these studies of 117 Helianthus species (Fig. 1, Table S1, P>0.05 for all). The nutrient limitation was substantial 118 enough to significantly reduce vegetative shoot biomass in all six studies (  (Fig. 3). 141

Variation in g night and E night nocturnally, across leaf lifespan and plant reproductive stages 143
A 24-hour time course was measured for H. annuus in Fall 2005-2. g day , E day and 144 photosynthesis showed typical patterns, increasing rapidly in the morning and declining during 145 the afternoon. g night and E night , though low compared to daytime rates, increased through the 146 night in the sufficiently-watered plants despite a small increase in atmospheric vapor pressure 147 deficit (VPD a ) though the night (Fig. 3) Table S1). Pre-reproductive plants (5.5 weeks old) had higher g night and E night than did 174 reproductive plants (10 or 15.5 week old). Plant reproductive stage also affected photosynthesis, 175 which was higher in pre-reproductive plants (F 2,46 =6.69, P<0.01), but not g day and E day (P>0.05). 176 The contribution of g cuticular to g night 178 During the Fall 2004-1 and Spring 2005 studies g cuticular , functionally defined as water loss 179 though the cuticle with stomata at maximal closure, was measured on excised, wilted leaves. In 180 Fall 2004-1 g night (total leaf conductance to water at night) was higher than g cuticular for all four 181 wild Helianthus species (Fig. 5). In Spring 2005, g night was again higher than g cuticular (Fig. 5). In 182 both studies g cuticular measured on leaves was higher than instrument error (P<0.001), which 183 averaged -7.5 x 10 -6 mol m -2 s -1 during g cuticular measurements. During Spring 2006, g cuticular was 184 measured on intact leaves of plants infused with exogenous ABA into the xylem. g cuticular was 185 lower than g night measured on intact leaves of control plants for both wild H. annuus and 186 domesticated H. annuus (Fig. 5). 187 Looking across all three studies, g cuticular for wild H. annuus ranged from 0.013 to 0.023 mol 188 m -2 s -1 and there was good agreement between measures made with the two different techniques 189  (Table S1), and the g night values were relatively large and greater than explained 199 by g cuticular . 200 In the Fall 2005-2 study, gravimetric measures were compared to instantaneous measures 201 of transpiration. The gravimetric measures were approximately four-fold lower, reflecting their 202 integration over the entire night or day period, whereas instantaneous measures were timed to 203 capture maximal E night and E day rates. However, there was a strong correlation between the two 204 measurement techniques. Additionally, the percentage total E night of total E day measured 205 gravimetrically over the 24-hours gave an estimate of 6%, which agreed well with the 5% 206 estimate from instantaneous gas exchange measures during the same day/night period. This 207 added validity to our estimates based on instantaneous measures. 208 209

Response of g night and E night to soil nutrient and water manipulation 210
We hypothesized that regulation might occur for increased g night under limited nutrient 211 conditions to increase bulk flow of soil solution to the roots and reduce the development of a 212 nutrient depletion zone in the rhizosphere. Although the soil nutrient limitations were sufficient 213 to limit shoot and reproductive biomass and generally to reduce leaf nitrogen concentration, they 214 did not affect g night and E night in any of the than H. petiolaris, consistent with the direction predicted by selection for higher g night in lower 234 nutrient habitats, but the magnitude of difference was relatively small and appeared largely 235 driven by greater g cuticular in H. deserticola. 236 g night and E night did decline in response to water limitations that were generally sufficient 237 to decrease leaf predawn xylem pressure potential, g day , E day and photosynthesis. Declines were 238 such that g night in the limited water treatments was generally within the range we recorded for 239 functionally defined g cuticular . For three of the four studies, the water limitation was short term 240 and consisted of withholding water just prior to measurements on fully mature leaves, so that the 241 effect on g night could not be due to a long term change in leaf structure, stomatal density or size, 242 or cuticle. The decline in g night and E nigh due to water limitation demonstrates that guard cell 243 regulation of nighttime water loss is possible, analogous to daytime regulation of water loss in 244 response to soil drying. Our results agree with previous results showing lower g night associated 245 with decreased plant water status in Hibiscus cannabinus (Muchow et al., 1980), Pseudostuga 246 menziesii (Running, 1976;Blake and Ferrell, 1977) and To generalize across plant life stages we investigated variation in g night and E night across 286 plant reproductive stages, controlling for leaf age. Pre-reproductive H. annuus showed 287 significantly higher g night and E night than individuals that were flowering or setting seeds. This 288 trend was not mirrored in daytime rates. Our results are consistent with those of Grulke et al. 289 (2004) who found g night to be higher in large saplings compared to mature ponderosa pine. 290 Young plants, during rapid vegetative growth, expend a large portion of respiratory energy 291 on nutrient uptake and this proportion generally declines as plants age (Marschner, 1995  Although our tests of g night responses to nutrients and water occurred within each study, 317 and cross study comparisons were not preplanned, the study differences in maximum g night 318 deserve some comments. For wild H. annuus in the nutrient and water manipulation studies, 319 g night of sufficiently-watered plants ranged from 0.04 to 0.12 mol m -2 s -1 (Figs. 1-2, Table S1). 320 Because studies were conducted in different seasons and years, some of the variation may have 321 been due to differences in the growth environment, and to VPD l differences during the nights 322 and days of gas exchange measurements. However, the study with the lowest g night (Fall 2005-1) 323 did not stand out as having the highest VPD l on the night or accompanying day of gas exchange 324 measurements, or an unusual VPD a across the growth interval of the study. It is possible that 325 using study means obscures a specific time interval where VPD a affected leaf development and 326 exploration of growth environment (temperature, humidity, CO 2 levels, light quantity and 328 quality, plant nutritional status, growth medium, etc.) on leaf structure, stomatal density and size, 329 cuticular properties, and maximum g night (Hetherington  grown in a greenhouse with natural daylight supplemented to 12 to 14 hours with metal-halide 361 lamps. Temperatures were generally set to be at or above 26ºC (day) and 16ºC (night). For the 362 six studies that had greenhouse weather available for the growth interval (Fall 2004-1, Fall2004- The sufficient and limited nitrate Hoagland solutions contained equal amounts potassium (176 372 µg mL -1 K) and phosphorus (31 µg mL -1 P). Additional macronutrients were calcium (50 µg 373 mL -1 in high; 10 µg mL -1 in low), sulfur (8 µg mL -1 in high; 120 µg mL -1 in low) and magnesium 374 (55 µg mL -1 in high; 6 µg mL -1 in low). For both chambers air temperature was set to ambient and CO 2 was supplied at 400 µmol 401 mol -1 . Flow was set to 125 to 200 µmol s -1 at night and 700 µmol s -1 during the day. Chamber 402 fan speed was set to high. To partially compensate for removal of the boundary layer due to the 403 chamber mixing fan, chamber relative humidity was manually manipulated to a target 5-10% 404 above ambient (assessed with open chamber). The standard chamber directly measures leaf 405 temperature and before every set of measurements the leaf thermocouple was checked to ensure 406 it was reading accurately to within 0.1 o C. Sample and reference IRGAs were matched prior to 407 every plant for nighttime measurements. Measurements were also made with an empty chamber 408 or with dry paper in the chamber every four to six leaf measures to assess instrument error. 409 Averaged by study, estimates of instrument error obtained with the standard or Arabidopsis 410 chamber at night yielded values for g from 0.001 to 0.016 mol m -2 s -1 , which was always 411 substantially lower than plant measures. Plant measures were logged when readings were stable 412 and typically within 1-2 minutes of clamping onto the leaf. 413 Whenever possible leaves were chosen that would fill the leaf chamber (6 cm 2 for standard 414 chamber; 0.8 cm 2 for Arabidopsis chamber). When leaves that did not fill the chamber were 415 used, all leaves in the measurement set (including those that filled the chamber) were marked 416 before removal from the chamber to indicate placement of the chamber gaskets. The following 417 day gas exchange leaves were cut to remove all area that was not inside the chamber and scanned 418 (Winfolia, Regent Instruments Inc., Quebec, Canada) to determine area. Leaves that did not fill the chamber were used in the Arabidopsis chamber in Fall 2004-1 (minimum area 0.45 cm 2 ) and 420 in the standard chamber in Fall 2005-1 (minimum area 4.5 cm 2 ). 421 Daytime measurements were typically made between 9 am and 2 pm and nighttime 422 measurements were typically made between 1 am and the beginning of astronomical twilight 423 (sun 12 0 below the horizon). Measures made at three times spaced though the night confirmed 424 that this period captured maximum g night but was well before a predawn stomatal opening would 425 occur. 426 In the Fall 2005-2 study nighttime water loss was measured both instantaneously using the 427 LI-6400 as well as gravimetrically. Gravimetric measures of transpiration made over a 24-hour 428 time span were achieved by sealing the pot and root system in a bag, bagging all flower heads 429 and weighing at the beginning and end of the day and night periods. To obtain water loss per 430 area, all leaves were harvested the following day and total leaf area was measured using a LI-431 3100 leaf area meter (LiCor, Lincoln, Nebraska, USA). 432 433

Assessment of cuticular water loss 434
Cuticular conductance (g cuticular ) was defined functionally as conductance through the 435 cuticle and stomata at maximum closure induced by either leaf wilting (water stress) or 436 exogenous ABA application. As such it includes both water loss through the cuticle and water 437 loss through stomata at minimum aperture. The conductance provided by the LI-6400 (g night or 438 g day in this study) is a total of both g cuticular and g stomata in parallel. Stomatal conductance at night 439 was calculated as g night minus g cuticular (Nobel, 2005). Experiments were either complete randomized block designs or completely randomized 476 (Table 1). When gas exchange measurements were made across several days and nights, plants 477 were grouped by block so that random effects due to night of measurement (e.g.  Table 2: Vegetative shoot biomass at harvest and total leaf nitrogen (N) content of gas exchange leaves for studies that included a nutrient limitation treatment. If no treatment is designated (i.e. "---") then all plants in that study received sufficient levels of that resource. Values are lsmeans ± 1 SE. F-values and associated degrees of freedom (F df num, df denom. ) are presented for each model effect (PROC MIXED ANOVA, block as random). F-values in bold indicate statistical significance (* P<0.05, ** P<0.01, *** P<0.001).

Nutrient treatment
Water treatment shoot (g) N (mg g -1 ) for gas exchange leaf Model Effects Figure 1: Effect of manipulating soil nutrient availability on nighttime leaf conductance (g night ) showing all of the tests for wild Helianthus annuus. In Fall 2003-1 availability of all macro-and micronutrients were manipulated, whereas only nitrogen, available as nitrate, was manipulated in the additional four studies. Bars are lsmeans ± 1 SE. See Table S1