Practical implications of different phenotypic and molecular responses of evergreen

1 conifer and broadleaf deciduous forest tree species to regulated water deficit in a 2 container nursery 3 4 Piotr Robakowski, Tomasz Wyka, Wojciech Kowalkowski, Władysław Barzdajn, Emilia 5 Pers-Kamczyc, Artur Jankowski, Barbara Politycka 6 7 8 Piotr Robakowski, Poznan University of Life Sciences, Faculty of Forestry, Wojska 9 Polskiego 71E, PL 60-625 Poznań, Poland, pierrot@up.poznan.pl, ORCID: 0000-0001-556410 7360 11 Tomasz Wyka, Adam Mickiewicz University, Faculty of Biology, General Botany 12 Laboratory, Umultowska 89, PL 61-614 Poznań, twyka@amu.edu.pl; ORCID: 0000-000313 0569-8009 14 Wojciech Kowalkowski, Poznan University of Life Sciences, Faculty of Forestry, Wojska 15 Polskiego 71E, PL 60-625 Poznań, Poland, wojciech.kowalkowski@up.poznan.pl, ORCID: 16 0000-0002-5801-9818 17 Władysław Barzdajn, Poznan University of Life Sciences, Faculty of Forestry, Wojska 18 Polskiego 71E, PL 60-625 Poznań, Poland, barzdajn@up.poznan.pl, ORCID: 0000-000219 0018-2057 20 Emilia Pers-Kamczyc, Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, PL 21 62-035 Kórnik, Poland, epk@man.poznan.pl, ORCID: 0000-0002-5610-2124 22 Artur Jankowski, Adam Mickiewicz University, Faculty of Biology, General Botany 23 Laboratory, Umultowska 89, PL 61-614 Poznań; Institute of Dendrology, Polish Academy of 24 Sciences, Parkowa 5, PL 62-035 Kórnik, Poland, artur.jankowski@amu.edu.pl, ORCID: 25 0000-0001-6765-8008 26 Poznan University of Life Sciences, Faculty of Horticulture and Landscape Architecture, 27 Department of Plant Physiology, Wołyńska 35, PL 60-625 Poznań, Poland, 28 barbara.politycka@up.poznan.pl, ORCID: 0000-0002-5078-6596 29 30 Author for correspondence, e-mail: piotr.robakowski@up.poznan.pl, phone: +48 61 848 77 31 38 32 33 34 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 9 August 2020 doi:10.20944/preprints202008.0224.v1


Introduction
phase of growth, seedlings are irrigated every two days with 5 mm for P. sylvestris and A. 200 alba and with 7 mm every three days for the broadleaved deciduous species. In our 201 experiment, we had regard for the norms and nurseryman' experience. Four daily irrigation 202 levels were established: 6 mm (6 l/m -2 , 100%), 4.5 mm (4.5 l/m -2 , 75%), 3 mm (3 l/m -2 , 50%) 203 and 1.5 mm (1.5 l/m -2 , 25% of the maximal dose). When recalculated per area of one cavity of 204 container, 12 (100%), 9 (75%), 6 (50%) or 3 ml (25%) of water were delivered every day. 205 After leaf expansion, water amount reaching the substrate in cavity was reduced when 206 compared to these values due to interception. To compare species-specific responses to the 207 irrigation doses, we applied the same irrigation regime for each species. Each irrigation 208 regime was replicated four times with four containers per species and irrigation treatment per The terms water deficit and drought have often been considered synonyms although water 229 deficit is more commonly used when referring to water availability below field capacity, 230 especially in relation to crop cultivation whereas drought is generally related to low 231 precipitation over a period of time. In our study, seedlings were cultivated under controlled conditions in an unheated tunnel, therefore we use the term 'water deficit' for irrigation levels 233 below the established norm. 234 Plants were watered by an overhead sprinkler system. The different irrigation levels 235 were obtained by regulating the duration of water delivery over particular blocks. The water 236 pressure (0.25 MPa in each sprinkler) and irrigation time were monitored using an automated 237 system (Rathmakers, Germany). A pluviometer installed below the overhead sprinklers were 238 used to measure water amount in millimeters in each irrigation treatment.

240
Growth, biomass allocation and water contents 241 The height and root collar diameter of seedling were measured after 60 days of growth under 242 experimental conditions (n = 10, nnumber of seedlings per block, species and per 243 treatment). Seedlings were subsequently removed from containers and their roots were gently 244 washed (n = 4). Plants were dissected into roots, stem and leaves and fresh mass (FM) of each 245 fraction was determined. Biomass fractions were then dried at 65º C for 7 days until the weight 246 was unchanged using a climate cabinet (Pol-Eko, Poland), and weighed for dry mass 247 determination. Water amount in each type of organs was calculated by subtracting organ dry 248 mass from its fresh mass and water content per unit of dry mass was obtained by dividing the 249 water amount by dry mass of the organ fraction or by total seedling dry mass. On 13 August 2015 one mature, undamaged leaf per seedling was sampled from eight 253 seedlings (two from each block) in 25%, 50% and 100% irrigation regimes. We expected that 254 anatomical acclimation in needles could be observed among extremely different irrigation 255 treatments, therefore the 75% irrigation treatment was not considered in the microscopic leaf 256 variables. A 2 mm wide segment from the central part of the needle or the broadleaf lamina 257 (including midrib) was excised and fixed by vacuum infiltration for 2 hours in a mixture of 258 paraformaldehyde (2%) and glutaraldehyde (2%)  Whatman # 2 filter paper. A 2 ml aliquot of filtrate was incubated with 2 ml of acid-ninhidrin 279 and 2 ml of glacial acetic acid in a test tube for 1 hour at 100°C, and the reaction was 280 terminated in an ice bath. The reaction mixture was extracted with 4 ml toluene by mixing 281 with a test tube stirrer for 15-20 sec. The chromophore containing toluene was aspirated from 282 the aqueous phase, warmed to room temperature and the absorbance read at λ = 515 nm 283 (Spekol, CarlZeiss, Jena) using toluene as blank. The proline concentration was determined 284 from a standard curve and calculated on a dry mass basis using the formula Sk= K × A × T, 285 where: K is coefficient calculated with the standard curve, A is absorbance and T is 286 conversion factor accounting for the volume of toluene. Proline leaf concentration was 287 expressed in µg g -1 DM.   Table 1). Real-time quantitative RT-PCR (qRT-PCR) was used to 320 describe plants responses to water deficit. On the base of published data, we selected drought-321 related genes which were previously found by others to be related to drought stress response 322 (up-regulated or down-regulated) or to abiotic stress in analyzed species. In F. sylvatica, 323 abscisic acid (ABA)-related drought signaling genes were analyzed: NCED1 -e9-cis-epoxy-  Prior to analyses, the data were tested for normality and homogeneity of variance in groups 349 with Shapiro-Wilk's and Levene's test, respectively. Data were log10 transformed to fulfill the 350 ANOVA conditions. One-way ANOVA with irrigation regimes as the fixed factor and blocks 351 as the random factor was applied to compare the irrigation treatments within each species for 352 water contents in organs, whole plant dry mass, biomass allocation and gene expression or two-353 way ANOVA with species and irrigation regimes as fixed factors for leaf proline concentration 354 and anatomical leaf traits at P < 0.05. When ANOVA showed significant differences, the mean 355 values were compared with the analysis of contrasts at P < 0.05.

358
Seedling water status 359 At time of harvest both gymnosperm species (Abies and Pinus) had higher LWC (leaf water 360 content) and lower RWC (root water content) than the angiosperm species (Fagus and Quercus) 361 whereas their SWC (stem water content) were similar (Fig. 1). Lower irrigation resulted in 362 slightly reduced LWC in Quercus and Pinus although most pairwise contrasts were non-363 significant ( Fig. 1 a). There were also slight decreases of RWC in Fagus and SWC in Quercus and Abies due to reduced irrigation ( Fig. 1 b, c). Thus, the measurement of water contents 365 indicated only moderate organ-level water stress.

367
Growth 368 Reduction in water availability resulted in decreased height and root collar diameter of Fagus, 369 Quercus and Pinus seedlings while in Abies the growth response to the different irrigation 370 regimes was not significant (Table 1). The magnitude of response was largest in Fagus (2.2 and 371 1.8-fold reduction in respectively, height and diameter), followed by Quercus (1.8 and 1.5-fold 372 reduction and Pinus (1.5 and 1.2 fold). Significant height reductions occurred as irrigation was 373 decreased from 100% to 75% (Fagus and Quercus) and from 75% to 50% and from 50% to 25% 374 of the recommended dose (all three species). Mean slenderness indices (h/d) decreased with 375 reduced irrigation in Fagus, Quercus and Pinus but not in Abies (Table 1).

376
Biomass accumulation differed significantly among species, with seedlings of the two 377 angiosperms Fagus and Quercus reaching over 2.5 g dry mass under full irrigation, in contrast 378 to the gymnosperms Abies and Pinus that reached only, respectively, 0.1 and 0.4 g ( Table 2; 379 Fig. 2a, b). Irrigation regime affected seedling growth in a dose-response manner in three out 380 the four species (Fig. 2). Under 25% irrigation, dry mass of Fagus seedlings was over 5 times 381 smaller than under the 100% dose, whereas in Quercus the difference was about three-fold ( Fig.   382 2a). There was, however no effect of varied irrigation on the biomass of Abies and the biomass 383 decrease between 100% and 25% doses in Pinus was approximately by 1/3 (Fig. 2b). Alteration of biomass allocation in response to reduced irrigation was species-specific ( Fig. 3 387 a-d). The strongest response was observed in Pinus, in which ABMR (aboveground to 388 belowground mass ratio) strongly declined with irrigation reduction from 75 to 50%, resulting 389 from an increased RMF (root mass fraction) and decreases in both LMF (leaf mass fraction) 390 and SMF (stem mass fraction). In contrast, allocation in Abies was not affected by irrigation. In 391 Fagus, reduced irrigation resulted in increased LMF and decreased SMF without effect at the 392 root or ABMR level. Finally, in Quercus only a minor decrease in ABMR was noted at 393 irrigation reduction from 100 to 75%, with variability in organ-level allocation not unaccounted 394 for by irrigation. The expression levels of selected stress-response genes 416 In F. sylvatica, all transcripts of the analyzed genes were present in leaf samples, but their 417 relative level of gene expression was not affected by treatment (P > 0.05). In Q. petraea, 418 efficiency of primers for TUB was more than 2.0, therefore this transcript was not analyzed. An

Species-specific growth responses to irrigation and alterations in gene expression
In this study of containerized-seedlings, large interspecific differences were observed in 431 the response of seedling growth to reduced irrigation. These differences were associated with 432 the classification of the different species. In agreement with our first hypothesis, the broadleaf 433 deciduous genera, Fagus and Quercus, exhibited a greater magnitude of response to reduced 434 irrigation than the two evergreen, conifer genera, Abies and Pinus. Within the deciduous species, 435 F. sylvatica was more greatly impacted by reduced water availability than Q. petraea. The be the result of their contrasting growth phenology as seedlings. The slow-growing species, A. 445 alba, with its small amount of leaf biomass, requires less water than the faster growing, pioneer 446 species, P. sylvestris. Although all seedlings were exposed to drought during their rapid-growth 447 phase, growth in Abies is naturally terminated sooner than in Pinus; thus the ability of Abies to 448 respond to the decreased water availability may be restricted by its phenological cycle. This are also heavier than seeds of P. sylvestris and thus provide more resources to germinating 452 seedlings during their initial growth than seeds of P. sylvestris. This would provide an 453 advantage to A. alba seedlings, especially under water deficit conditions [76]. The interspecific 454 differences observed in our experiment resulted to some extent from a different species-specific 455 interception, which reduced amount of water reaching roots of seedlings growing in containers.

456
In mature P. sylvestris stands in Germany, mean precipitation interception was 32% [77], and 457 in leafed period in F. sylvatica forest in Belgium, the level of interception was 28% [78]. These 458 data cannot be directly compared with the interception of our study seedlings, but they allow to 459 estimate the importance of interception in the water balance of trees under natural conditions.

460
In our study, however, the interception effect on water amount reaching the substrate and roots 461 of seedlings in containers was substantially reduced: (1) Water was delivered at high pressure: 462 at the sprinklers the pressure was 4 bars, and at crowns of seedlings around 3.0 -3.2 bars and never lower than 2.5 bars. Due to high pressure, even after full leaf expansion, water reached 464 the substrate in cavities; (2) We cultivated our plants from seeds, thus at the beginning of the 465 experiment, the value of interception was 0 and during seeds' germination and initial growth, 466 it did not substantially affect the amount of water irrigating the substrate. An effect of 467 interception was important at the end of our experiment, after full leaf expansion. However, 468 this effect of different interception might be at least partially compensated by the high pressure 469 of irrigation water, high air humidity in the tunnel and foliar water absorption of seedlings 470 which can be significant under water deficit [79].

471
The select genes allowed us to compare tolerance to reduced irrigation within each of LEA expression observed in that study in all parts of the seedlings indicates that the level of 491 drought stress imposed on P. sylvestris seedlings in that study was higher than the level of 492 drought stress imposed in the present study. A lack of significant expression or low expression 493 of the selected stress related, potential marker genes in the studied species suggests that the 494 water deficit stress was efficiently moderated by stomatal closure.

Seedling water status
Relative tissue water content was used as a measure of plant water status. In our study, 498 organ-level water content decreases caused by water deficit were small especially in the 499 conifers, suggesting an efficient control of transpirational water loss. This was especially true 500 in Abies. In addition, seedlings' water status could be substantially improved by foliar 501 absorption of intercepted water, especially under water deficit. In an earlier study, the 502 substantial improvement of water status, exceeding 1.0 MPa water potential for drought-503 stressed Juniperus monosperma plants was observed, following precipitation on an 504 experimental plot that excluded soil water infiltration [79].

505
Conifers at the same time showed much larger hydration of leaves across irrigation 506 levels as compared to the two angiosperms. This may be attributed to the low amount of 507 mechanical tissues and the large mesophyll content in juvenile needles. Interestingly, conifer 508 root systems were much less hydrated than angiosperms, however since conifer roots 509 constituted lower fraction of biomass than leaves, conifer seedlings held more water per gram 510 total biomass than the angiosperm seedlings.

511
Our results suggest that the ability to accumulate and conserve foliar water may 512 constitute a fundamental difference between drought-survival strategies of broadleaved 513 deciduous and evergreen conifer species. This is supported e.g., by a tighter stomatal control, 514 substantial decrease in sap flow rates and transpiration in P. sylvestris compared with more 515 drought-tolerant Quercus pubescens [84,85]. Our suggestion is consistent with the higher 516 growth rates of deciduous angiosperm seedlings achieved through a more intensive gas 517 exchange, especially when expressed on the leaf mass basis. In the general scheme of ecological 518 strategies, conifers appear to emphasize water conservation in contrast to specialization towards 519 water acquisition and spending in seedlings of broadleaf angiosperms.     Fagus F 3,60 = 0.48 n.s. Quercus F 3,60 = 7.00 *** Abies F 3,60 = 0.60 n.s. Pinus F 3,60 = 7.28 *** Fagus F 3,60 = 3.93 * Quercus F 3,60 = 1.59 n.s. Abies F 3,60 = 0.60 n.s. Pinus F 3,60 = 1.39 n.s.