Introduction

Leaf photosynthetic functions are key traits for the determination of tree growth. Enhancement of leaf photosynthesis will thus be a possible measure for improvement in the productivity of forest trees. Eucalyptus species are one of the most widely-planted multipurpose tree species in the world1,2, which are used as a source of paper pulp, wood, and timber. Eucalyptus species have a high growth rate and high photosynthetic rate, while soil water availability strongly limits their growth3,4; therefore, improvement in leaf photosynthesis and tree growth, as well as raising drought tolerance under limited water resources, are important issues for improving the production of Eucalyptus trees.

One of the promising measures to improve leaf photosynthesis is to modify the leaf mesophyll anatomy. Leaf photosynthesis is limited by (1) CO2 diffusion through the stomata, (2) CO2 diffusion through the mesophyll, and (3) photosynthetic biochemistry, in which both CO2 diffusion through the mesophyll and photosynthetic biochemistry are strongly affected by the leaf mesophyll anatomy, in particular the number of chloroplasts5,6,7,8. Thick and dense leaf mesophyll tissue generally has a high number of chloroplasts and thus involves high photosynthetic functions through high mesophyll CO2 diffusion and a large amount of carboxylation enzymes within a single species9,10,11 and among different species of angiosperms and ferns8. Furthermore, the above alterations in the leaf mesophyll anatomy also potentially improve both the growth and drought tolerance of tree leaves through improvement in the water-use efficiency, when an increase in water-use efficiency is not accompanied by a concurrent decline in CO2 fixation limited by mesophyll or photosynthetic biochemistry12. Although a high photosynthetic rate generally corresponds to high water loss from the stomata due to large stomatal conductance and high hydraulic conductance among different species13, an increase in the photosynthetic rate with little change in stomatal conductance has been reported in Eucalyptus leaves overexpressing radish aquaporin RsPIP2;114.

In this study, we focused on EcHB1 from Eucalyptus as a candidate for improving tree photosynthesis, growth, and drought tolerance through changes in the leaf anatomy. EcHB1 is a Eucalyptus camaldulensis homolog of the homeobox protein HAT22 of Arabidopsis thaliana (AT4G37790). They belong to the HD-Zip II transcription factor family which contains a homeodomain and leucine zipper motif. EcHB1 was originally isolated as a factor that controls the expression of multiple genes encoding enzymes for cell wall biosynthesis pathways15. In our previous study15, we generated Eucalyptus plants overexpressing EcHB1 under the control of a CaMV 35 S promoter, and reported an increase in the plant growth and alterations in flower morphology that suggest functional roles of EcHB1 on the cell growth in multiple plant organs including the leaves. In fact, we obtained significant alterations in leaf morphology in Eucalyptus trees overexpressing EcHB1 in our preliminary experiment. HD-Zip transcription factors regulate plant growth responses through suppression or promotion of cell multiplication, differentiation, and expansion through phytohormone-regulated networks16,17. Previous genetic modification efforts for improvement in the growth and production of trees have focused on cellulose modification, imparting tolerance to biotic and abiotic stresses and modification of lignin content18, in which a decrease in the lignin content through the suppression of some genes, including Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and caffeate/5-hydroxyferulate O-methyltransferase (COMT), has been studied19,20,21. However, adequate lignification is essential for mechanical support and water transport in trees; the down-regulation of two genes encoding two 4CLs causes increased xylem vulnerability to embolism and decreased tree growth of poplar22.

The aims of the present study are to test the hypothesis that overexpression of EcHB1, an HD-Zip II transcription factor from Eucalyptus camaldulensis, improves the leaf photosynthetic function, growth, and drought tolerance through alterations in the mesophyll anatomy in Eucalyptus trees. Alterations in the following three factors: CO2 diffusion through the stomata, CO2 diffusion through the mesophyll, and photosynthetic biochemistry will be discussed as possible mechanisms explaining changes in leaf photosynthetic functions. Additionally, the number of leaves and growth in response to soil drought were examined for assessing alteration in the drought tolerance of Eucalyptus trees through overexpression of EcHB1.

Results

Gene expression levels and phenotypes

RT-PCR analysis showed that in Eucalyptus leaves, expression levels of EcHB1 in the overexpressed lines EcHB1-2 and EcHB1-10 were much higher than those in the control line GUT5 (14-23-fold, Fig. S1A). Northern blot analysis revealed that in the transgenic line EcHB1-2, EcHB1 was highly expressed in the meristem, leaf, upper xylem, lower xylem, and roots, while EcHB1 expression levels in these tree tissues were very low or almost negligible in the control line GUT5 (Fig. 1A).

Figure 1
figure 1

(A) Gene expression levels measured using Northern blot analysis of the meristem, leaf, upper xylem, lower xylem, and root of the control GUT5 and EcHB1-overexpressed line EcHB1-2, in which actin2 of Eucalyptus camaldulensis (EcACT2) was used as the control (see Supplemental Information). (B) Phenotypes of the control (GUT5) and EcHB1-overexpressed lines (EcHB1-2 and EcHB1-10), grown for 12 months. (C) Tree growth traits such as stem diameter, tree height, growth rate in stem diameter and tree height, shoot fresh weight (FW), root FW and leaf number. Blank, gray, and filled bars indicate GUT5, EcHB1-2, and EcHB1-10, respectively. Means with standard errors are shown for 3–12 trees. Differences between lines were analyzed using one-way ANOVA, in which different letters indicate that the difference was statistically significant (P < 0.05).

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Alterations in some of the tree phenotypes were obtained for the two transgenic Eucalyptus lines EcHB1-2 and EcHB1-10 compared to the control line GUT5 (Fig. 1B). In the overexpressed lines, tree heights and their growth rate were 1.5-1.6-fold, while diameters at the base of the stems were 0.7-fold and the root fresh weights were 0.4-0.6-fold those of the GUT5 (Fig. 1C). The shoot and stem fresh weights, stem growth rate, and leaf number were not changed in the overexpressed lines.

Leaf and photosynthetic traits

Significant alterations were again observed in the leaf morphological and anatomical traits in the transgenic lines EcHB1-2 and EcHB1-10 (Fig. 2A). These transgenic lines had densely-packed small leaves with increased number of chloroplasts; in these transgenic lines, the leaf area was 0.5-fold, while the mesophyll fractions (ratio of mesophyll tissues in leaf section), number of mesophyll cells, and chloroplasts was 1.3-1.9-fold of the control line GUT5 (Fig. 2B).

Figure 2
figure 2

(A) Images of the leaves of Eucalyptus and their transverse sections. (B) Leaf area, leaf mesophyll fraction, number of mesophyll cells and chloroplasts, in which means with standard errors are shown for 5–18 fully-expanded mature leaves. Blank, gray, and filled bars indicate GUT5, EcHB1-2, and EcHB1-10, respectively. Differences between lines were analyzed using one-way ANOVA, in which different letters indicate that the difference was statistically significant (P < 0.05).

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As a result of the dense mesophyll tissues and increased number of chloroplasts, the transgenic lines EcHB1-2 and EcHB1-10 had a high area-based photosynthetic capacity compared to the control line GUT5 (Fig. 3A). The maximum photosynthetic rate (Amax), maximum Rubisco carboxylation rate (Vcmax), and photosynthetic electron transport rate (J) in the transgenic lines EcHB1-2 and EcHB1-10 were 1.3-1.7-fold those of the control line GUT5 (Fig. 3B).

Figure 3
figure 3

(A) Photosynthetic light response curves and A-Ci curves in the leaves of Eucalyptus. (B) Photosynthetic parameters such as maximum photosynthetic rate (Amax), maximum carboxylation rate (Vcmax), and maximum electron transport rate (J) estimated from light response curves and A-Ci curves in the leaves of Eucalyptus. Means with standard errors are shown for 4–12 fully-expanded mature leaves. Blank, gray, and filled bars or symbols indicate GUT5, EcHB1-2, and EcHB1-10, respectively. Differences between lines were analyzed using one-way ANOVA, in which different letters indicate that the difference was statistically significant (P < 0.05).

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We determined the CO2 diffusional limitations during photosynthesis in the Eucalyptus leaves (Fig. 4). Stomatal conductance (gs) in the transgenic lines was slightly higher (1.2-fold) or similar to that in the control lines, while mesophyll conductance (gm) in the transgenic lines EcHB1-2 and EcHB1-10 was 1.8-1.9-fold that of the control line GUT5 (Fig. 4A). Although the stomatal and biochemical limitations were similar between the lines, the mesophyll limitations of leaf photosynthesis were smaller in the EcHB1-10 line compared to the control line GUT5 (Fig. 4B). The higher gm in the transgenic lines EcHB1-2 and EcHB1-10 correlated to a higher chloroplast surface area exposed to intercellular airspaces (Sc), and the higher photosynthetic rate at 1500 µmol m−2 s−1 of PPFD (A1500) in the transgenic lines correlated to a higher gm (Fig. 4C).

Figure 4
figure 4

(A) Stomatal conductance (gs) or mesophyll conductance (gm) estimated using the isotope method or the curve-fitting method in the leaves of Eucalyptus. The data for EcHB1-10 obtained by the isotope method were eliminated because of the unexpected effect of senescence. (B) Photosynthetic limitations of the stomata, mesophyll, or biochemical traits. The differences between the control and EcHB1-overexpressed lines were tested using using the t-test (isotope method) or one-way ANOVA (curve-fitting method), with different letters indicating a statistically significant difference (p < 0.05). Means with standard errors are shown for 4–12 fully-expanded mature leaves. (C) Relationship between the surface area of chloroplasts facing intercellular airspaces (Sc) and gm, and between gm and the photosynthesis rate at 1500 μmol m−2 s−1 of PPFD (A1500). Means with standard errors for individual trees are plotted. Blank, gray, and filled bars or symbols indicate GUT5, EcHB1-2, and EcHB1-10, respectively.

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The data for photosynthesis and leaf morphological or anatomical traits were pooled for the two transgenic lines that had high EcHB1 expression levels (EcHB1-2 and EcHB1-10), and then compared to those of the control line GUT5 (Table 1). Leaf photosynthetic traits were increased in EcHB1-overexpressed lines except for stomatal conductance (gs), which corresponded to an increase in the anatomical factors enhancing CO2 diffusion (Sc, 1.6-fold) with no change in the cell wall thickness, which would inhibit CO2 diffusion. The increase in the number of mesophyll cells (1.4-fold) was accompanied by decreases in the palisade and epidermal cell width (0.8-fold), and an increase in the number of chloroplasts (1.5-fold) was followed by decreases in the width and thickness of the chloroplasts (0.8–0.9-fold). A higher leaf mass per area (LMA, 1.2-fold) while a similar mesophyll thickness was obtained. Stomatal traits including the stomatal size and density were similar between the EcHB1-overexpressed lines and GUT5.

Table 1 Mean values of photosynthetic, anatomical, and morphological traits of the 4–22 fully-expanded mature leaves in the 4-year-old trees.
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Principle component analyses were performed for the traits that were different between the EcHB1-overexpressed lines and GUT5 (Table 1), among the traits that were related to leaf photosynthetic functions (Fig. 5A), and among the leaf anatomical traits (Fig. 5B). The first main axis (PC1) is considered to be suitable for explaining variabilities in the data for both analyses, because PC1 accounted for 75–87% of the total variability in the data. Gas exchange parameters, such as Amax, gm, Vcmax, J, and A/E, had negative loadings, and the anatomical traits of Sc and LMA also had negative loadings, while the leaf area (LA) had a positive loading over PC1 (Fig. 5A). Overall, the size of the cells or chloroplasts had negative loadings, which corresponded to the negative loading of LA over PC1 (Fig. 5B). In contrast, the number of cells, number of chloroplasts, and fraction of mesophyll cells had positive loadings, with positive loadings of LMA and Sc. In these biplots, GUT5 plants had positive scores, while EcHB1-2 and EcHB1-10 had negative scores over PC1.

Figure 5
figure 5

Biplot of the principle component analyses for leaf and photosynthetic traits where the differences between the control and EcHB1-overexpressed lines were statistically significant (see Table 1). Mean values of traits for individual trees were used for the analysis (n = 5), in which open, gray, and filled circles indicate the principle component scores for GUT5, EcHB1-2, and EcHB1-10, respectively. Arrows indicate factor loadings of each trait. (a) Leaf traits and photosynthetic parameters such as leaf area (LA), leaf mass per leaf area (LMA), surface area of chloroplasts facing intercellular airspaces (Sc), maximum photosynthetic rate (Amax), maximum carboxylation rate (Vcmax), maximum electron transport rate (J), and transpiration efficiency (A/E). (b) Principle component analysis for the anatomical or morphological traits such as epidermal cell width and thickness (Epi-W, and Epi-T), palisade cell width (Pali-W), chloroplast width and thickness (Chls-W and Chls-T), LA, spongy cell length (Spg-L), number chloroplasts (Chls-num), mesophyll fraction and number of cells (Meso-fraction and Meso-cellnum), LMA, and Sc.

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Photosynthetic traits on a tree-basis and drought tolerance

The evaporation rate measured at 1500 μmol m−2 s−1 of PPFD (E1500) was smaller in the line EcHB1-2 than GUT on a tree-basis (0.6-fold) and on a leaf-basis (0.5-fold), while the line EcHB1-2 had a similar E1500 on a leaf-area basis to those in the line GUT5 (Table 2). On a tree-basis, photosynthesis measured at 1500 μmol m−2 s−1 of PPFD (A1500) and the surface area of the chloroplasts facing intercellular airspaces (Sc) were similar between the line GUT5 and EcHB1-2, although in the transgenic line EcHB1-2, these traits were higher and lower on a leaf-area-basis and a whole-leaf-basis, respectively.

Table 2 Photosynthesis rate and evaporation rate measured at 1500 μmol m−2 s−1 of PPFD (A1500, E1500) and surface area of chloroplasts facing intercellular airspaces (Sc) on a leaf-area basis, whole-leaf basis, and tree basis.
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Overall, the drought and following recovery experiment showed enhanced drought tolerance in EcHB1-2 (Fig. 6). The number of leaves was higher in EcHB1-2 than that in GUT5 at all three drought levels during the 4-month drought experiment, except for the 3-month high-drought level (Fig. 6A). The tree height was higher in EcHB1-2 than that in GUT5 at all three drought levels after a 4-month drought (Fig. 6B). Stem diameter was similar in EcHB1-2 and GUT5 at the low-drought level, and differences between EcHB1-2 and GUT5 at the middle- and high-drought levels became less distinct compared to before the drought experiment. After the 3-month recovery experiment, enhanced growth was observed overall in the transgenic line EcHB1-2, in which differences between the lines were more significant under the low-drought level (Fig. 6C). The ratios of tree height, shoot fresh weights and stem fresh weights of EcHB1-2 to those of GUT5 were 1.4-1.5-fold, 1.3-1.4-fold, and 1.2-1.4-fold at the low-, middle-, and high-drought levels, respectively.

Figure 6
figure 6

Growth response of the 8-month-old Eucalyptus trees to drought and the following re-watering treatment for the control (GUT5) and EcHB1-overexpressed line (EcHB1-2). Blank and gray symbols or bars indicate GUT5 and EcHB1-2, respectively. (A) Number of leaves during the 4-month drought experiment. (B) Tree height and stem diameter before and after the 4-month drought treatment. (C) Tree height and shoot and stem fresh weight after 3 months of re-watering. Means with standard errors are shown for 7–25 trees. The differences between GUT5 and EcHB1-2 were tested using the t-test, with asterisks indicating a significant difference with  *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. indicating the difference was not significant. P-values are shown when 0.05 < P < 0.1.

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Discussion

Significant enhancements in the area-based leaf photosynthetic functions were obtained in Eucalyptus trees overexpressing EcHB1 (Fig. 3). This is the first study showing the effect of an HD-Zip class II transcription factor on the leaf photosynthetic function. Leaf photosynthesis is limited by 1) biochemical activities and 2) CO2 diffusion through the stomata and mesophyll6,7,8. Overexpression of EcHB1 in the leaf induced significant alterations in the leaf mesophyll anatomy (Fig. 2, Table 1), which contributed to significant enhancements in the area-based leaf photosynthetic functions (Fig. 3) through enhancements in both the biochemical activities (Vcmax and J, Fig. 3) and CO2 diffusion through the mesophyll (gm, Fig. 4). The principle component analysis of leaf anatomical traits indicated that the marked increase in the mesophyll fraction (1.5-1.9-fold, Fig. 2B) was mainly due to the increase in the number of mesophyll cells (1.4-fold, Fig. 5B, Table 1). The increase in the number of mesophyll cells is related to an increase in the number of chloroplasts (1.5-fold, Fig. 5B, Table 1), which may induce enhancements in the photosynthetic biochemistry, e.g., the carboxylation enzyme Rubisco that regulates the velocity of carboxylation (Vcmax) and photochemical efficiency and the Calvin cycle that regulates the electron transport rate (J, Fig. 3B). When the relationship between the leaf anatomical traits and biochemical parameters was analyzed, a high Vcmax corresponded to a large number of chloroplasts in angiosperms and ferns8,23.

Overexpression of EcHB1 induced marked enhancement in the mesophyll diffusional conductance, gm (1.8-1.9-fold, Fig. 4A), which is strongly affected by alterations in the mesophyll anatomy (Fig. 5A); the significant increase in the number of chloroplasts (1.5-fold) with a decrease in chloroplast size (Table 1) resulted in a 1.6-fold increase in the chloroplast surface area (Sc, Table 1) for CO2 diffusion5. The concurrent increases in Sc and gm in the EcHB1-overexpressed lines (Fig. 4C), together with the concurrent increases in gm and A (Fig. 4C), supports the significant role of Sc for determining gm, and thus, photosynthesis. On the other hand, overexpression of EcHB1 induced little alteration in the stomatal conductance, gs (Fig. 4A). The stomatal density and size strongly affect stomatal conductance, gs24. Low stomatal density as well as large stomatal size reduces gs, because low stomatal density reduces the total stomatal pore area in leaves, while large stomatal size increases the diffusion path length25. The small change in stomatal conductance (gs) by the overexpression of EcHB1 (Fig. 4A) may be due to the lack of alterations in both the stomatal density and size (Table 1). CO2 diffusion through the stomata and mesophyll imposes almost comparable limitations in photosynthesis in different tree groups including deciduous, evergreens, and conifers7. The present study for Eucalyptus trees showed that the mesophyll tended to impose lower limitations in the overexpression lines EcHB1-2 and EcHB1-10 compared to GUT5 (Fig. 4B). This result reflects the enhancement of gm by the overexpression of EcHB1 (Fig. 4A).

Overexpression of EcHB1 improves the drought tolerance of Eucalyptus trees (Fig. 6). This improvement in drought tolerance may be related to the alteration in the transpiration of the whole tree. As a result of (1) an unchanged transpiration rate on a leaf-area basis, (2) a significant reduction in leaf area (0.5-fold), and (3) a small increase in the number of leaves (1.3-fold, data not shown), the estimated transpiration rate on a tree basis was reduced (0.6-fold) for the EcHB1-overexpressed lines (Table 2). This will reduce water loss from trees, and thus, may contribute to prevent the EcHB1-overexpressed lines from leaf-falling. Additionally, we observed alterations in the vessel anatomy in the stems of EcHB1-overexpressed lines, with a decrease in the radial diameter and a high cell wall thickness (data not shown). These changes will enhance stiffness of xylem cells but reduce the hydraulic conductivity in stems26,27, and as a result, will also reduce water loss from the trees during drought. Although the lower root biomass in the EcHB1-overexpressed lines (Fig. 1B,C) may cause less water uptake from the soil and thus potentially induce a sensitive response to drought28, the effect of reducing water loss (Table 2) may overcome the effect of less water uptake under the severe drought condition in the present study. The lower rate of leaf-falling during drought (Fig. 6A) may contribute the rapid recovery of the number of leaves after re-watering and thus improves the growth of the trees (Fig. 6C). Some morphological properties of the EcHB1-overexpressed lines on a tree-basis are similar to the Eucalyptus trees with high drought tolerance. Eucalyptus provenances from dry areas in Australia have a higher leaf per stem area ratio29, which supports our result of a larger number of leaves with a smaller stem diameter, i.e., a larger number of leaves per stem area, in the EcHB1-overexpressed line EcHB1-2 (Fig. 6A).

The HD-Zip I, HD-Zip II, and HD-Zip III transcription factor networks regulate plant growth responses to environmental conditions through suppression or promotion of cell multiplication, differentiation, and expansion through phytohormone-regulated networks16,17. Some Arabidopsis HD-Zip II genes are required for shade-induced hypocotyl elongation30,31, as well as controlling several auxin-regulated developmental processes including lateral organ polarity32. Overexpression of EcHB1, an HD-Zip II transcription factor, induced an increase in tree height, a decrease in tree diameter, and a decrease in root growth in Eucalyptus (Fig. 1B,C), which is in line with the results of a previous study30; in Arabidopsis seedlings, the increasing level of Arabidopsis HD-Zip II gene AtHB2, induces shifts from radial cell expansion to longitudinal cell expansion in the hypocotyl, and involves an inhibition in root growth. The increase in plant height by the overexpressing EcHB1 under the control of CaMV 35 S promoter was also reported in tobacco plants15. These results suggest that the functions of EcHB1 for plant growth are similar to those of AtHB2.

Alterations in leaf morphology by the overexpression of EcHB1 (Fig. 2, Table 1) again suggests similar functions of EcHB1 to those of some Arabidopsis HD-Zip II genes including AtHB2. The decreased leaf size in EcHB1-overexpressed Eucalyptus (Fig. 2) supports the results of previous studies which found that overexpression of Arabidopsis HD-Zip II genes, AtHB2, AtHB4, HAT1, HAT2, and HAT3 decreased leaf expansion in Arabidopsis30,33,34,35,36. The reduction in leaf expansion may be partly due to the reduction in the width of epidermal cells in the leaf-width direction30 (Table 1). In the present study, we also found increases in the proliferation of leaf mesophyll cells, as well as increases in the number of chloroplasts in the leaf-thickness direction in the Eucalyptus plants overexpressing EcHB1 (Table 1), which suggest additional functions of HD-Zip II genes in leaf development.

In conclusion, overexpression of EcHB1, an homeodomain leucine zipper (HD-Zip) II transcription factor, in the hybrid eucalypt GUT5 (Eucalyptus grandis and Eucalyptus urophylla) induced significant enhancements in leaf-area-based leaf photosynthesis. This improvement is due to both enhanced CO2 diffusion into chloroplasts and increased photosynthetic biochemical functions, which are affected by alterations in the mesophyll anatomy, e.g., an increased number of chloroplasts per unit leaf area. Overexpression of EcHB1 improved drought tolerance, which is mainly affected by the reduction in leaf area with no changes in stomatal morphology. Although negative regulations of leaf size by Arabidopsis HD-Zip II genes have been already reported, the positive regulation of cell division and number of chloroplasts by an HD-Zip II gene are new findings, which gave us insights into the role of the HD-Zip II gene.

Methods

Plant material

A hybrid Eucalyptus tree GUT5 generated from Eucalyptus grandis and Eucalyptus urophylla was used as the host plant14. This GUT5 is a selected clone that has a high ability to regenerate from a transformed callus. GUT5 shows good growth performance in subtropical areas, especially in Brazil where commercial forest plantations of Eucalyptus species are widely performed. Transgenic Eucalyptus GUT5 lines with a CaMV 35S promoter fused to EcHB1 cDNA (accession No. AB458829) were generated in our previous study15. We analyzed control (GUT5) and two transgenic lines (EcHB1-2 and EcHB1-10), in which 3–16 plants were used for the growth and drought experiment and one - three individual were selected for the detailed analysis of leaves. To obtain multiple transgenic Eucalyptus plants with the same genetic background, the stems of Eucalyptus plants were transplanted to 1.5-liter plastic pots filled with soil and then grown in temperature-controlled greenhouses of the Forestry Research Institute of the Oji Holdings Corporation (Kameyama, Japan) and Kyoto Institute of Technology (Kyoto, Japan). The plants were watered daily and fertilized biweekly with liquid fertilizer Hyponex (Hyponex Japan, Osaka, Japan) at a concentration of 1/1000. In the glasshouse of the Forestry Research Institute, the daily minimum and maximum temperatures ranged from 15–25 °C and 25–36 °C, respectively. In the glasshouse of the Kyoto Institute of Technology, the temperature was kept at 25 °C. Prior to the detailed leaf analysis, phenotypes of the plants and the growth rate were measured at the Forestry Research Institute for 12-month-old trees. The growth rates in stem diameter and tree height were calculated as increments of the values per 12 months.

Analysis of expression levels of EcHB1 in the transgenic lines of Eucalyptus trees

For RT-PCR analysis, RNA samples were extracted using an RNeasy Plant Mini Kit (Qiagen, CA). RT-PCR was performed using a PrimeScript RT-PCR kit (Takara Bio, Shiga, Japan) and EcHB1-specific primers (HB1RTF and HB1RTR)15. As an internal control to assess cDNA yield between the RNA samples, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers (5′-TAGCCATTTCCAGAACCCTCG-3′ and 5′-CGGAGATGACAACCTTCTTAG-3′)37 were used. The procedure of northern blot analysis including RNA extraction was as described previously15.

Measurement of leaf photosynthesis and stomatal and mesophyll conductance

Leaf photosynthesis was measured in the 4-year-old Eucalyptus trees, i.e., trees grown for four years since transformation at the Kyoto Institute of Technology, using the fully-expanded mature 5th to 10th leaves from the tops of the branches. The measurements were performed from August to October, in which four leaves from one each of individual trees of GUT5 and EcHB1-2 (n = 4), and four leaves from three individual trees of EcHB1-10 (n = 12) were used for the measurements. Photosynthetic parameters were measured using a photosynthesis system (Li-6400, Li-Cor, Lincoln, NE, USA), estimated from the photosynthesis (A)/intercellular CO2 concentration (Ci) curve fit model (A/Ci curve fit model)38,39 and the light response curve model40, with the leaf temperature and vapor pressure deficit (VPD) being set at 25 °C and 1.5 kPa, respectively. A/Ci curves and light response curves were obtained at a photosynthetic photon flux density (PPFD) of 1500 µmol m−2 s−1 and at 400 µmol mol−1 of ambient CO2, respectively, as in a previous study24. The photosynthetic rate and stomatal conductance at light saturation (A1500, gs-1500) were obtained at a PPFD of 1500 µmol m−2 s−1.

The mesophyll conductance (gm) of the leaves of the Eucalyptus trees was measured using two methods: the isotope discrimination method for six leaves (n = 6) and the curve-fitting method for four to twelve leaves (n = 4–12), as described previously8. For the isotope discrimination method, measurements were performed from November to December, in which the equations provided by the previous study that take the ternary effect into account41 were used for the estimation of gm. The data for EcHB1-10 obtained by the isotope discrimination method were eliminated from the analysis, because unexpected senescence occurred at the time of the measurement in the leaves of EcHB1-10. The second estimate of gm was obtained for all lines using the curve-fitting method38. The relative limitation of photosynthesis by stomatal conductance, mesophyll conductance, and biochemical capacity were estimated as in a previous study6.

Leaf morphological and stomatal properties

For measurements of the leaf morphological traits, fully expanded 5th to 10th leaves from the top of the branches were obtained during the same period as the photosynthesis measurements were performed (from August to October). Leaf transverse sections were obtained from five leaves (n = 5) at the central part of the lamina, and fixed and embedded in Spurr’s resin. Mesophyll anatomy was analyzed in 800-nm-thick transverse sections of leaves, observed using a light microscope (BX51-33, OLYMPUS, Tokyo, Japan), and the cell wall thickness of mesophyll cells was observed in 80-nm-thick transverse sections of leaves with a transmission electron microscope (JEM-1220, JOEL, Tokyo, Japan), as described previously42. Leaf anatomical traits were measured from digitized images of leaf sections of the lines GUT5, EcHB1-2, and EcHB1-10, using software43 (Image J). The surface area of chloroplasts exposed to the intercellular air spaces per unit leaf area (Sc) and mesophyll porosity were estimated as described previously using the images of light micrographs10. Briefly, Sc was estimated from the ratio of the perimeter length of chloroplasts/mesophyll cells exposed to the intercellular air spaces. Mesophyll fraction was calculated as 1 – mesophyll porosity. The number of mesophyll cells and chloroplasts, mesophyll thickness, mesophyll fraction, and Sc were measured from five images for each line (n = 5). Cell size (width and length) of the uppermost palisade mesophyll and the secondary layer of spongy mesophyll, the size of chloroplasts in palisade cells (width and thickness), and upper epidermal cell size (width and thickness) were measured in five cells of each image (n = 25). The thickness of the cell walls covered with chloroplasts was measured at two parts of the palisade cells in 10 images of sections of 80 nm thickness (n = 20) using a transmitting electron microscope (JEM-1220, JEOL Ltd., Tokyo, Japan). The chlorophyll content was estimated using a chlorophyll meter for six leaves (SPAD-502Plus, Konica-Minolta, Tokyo, Japan); the chlorophyll content was shown as SPAD values44. Leaf area was measured in four leaves using a scanner (Canoscan 9950 F, Canon, Tokyo, Japan) and image analysis software (Image J), and the leaves were dried at 60 °C for 48 h using an oven (MOV-112, SANYO Electric, Osaka, Japan) and weighed to assess the dry mass to calculate leaf dry mass per leaf area (LMA).

Stomatal density (SD) and stomatal size (length and width) were estimated in the lines GUT5 and EcHB1-10 using light micrographs of the replicas of leaf surface, as described in our previous paper24. The fully-expanded 5th leaves from the top of the branches were obtained in July, and SD was measured in two leaves of the four individuals (n = 8), and stomatal sizes were measured in five closed stomata of two leaves from the three individuals (n = 30).

Tree-basis photosynthesis and evaporation

The Leaf-area-basis photosynthesis rate and evaporation rate (A1500, E1500) measured at 1500 μmol m−2 s−1 of photosynthetic photon flux density (PPFD) and Sc were obtained for four leaves of the GUT5 and EcHB1-2 lines (n = 4), as described previously. Then, whole-leaf-basis A1500, E1500, and Sc were calculated using the averaged leaf-area-basis data multiplied by the leaf area of six leaves (n = 6). Tree-basis A1500, E1500, and Sc were calculated using the averaged whole-leaf-basis data multiplied by the number of leaves of the 8-month-old individual trees (n = 15–16).

Drought tolerance experiment

Seven to nine Eucalyptus trees of the lines GUT5 and EcHB1-2 were generated from tree cuttings or calli, and grown in 7.0-liter pots for eight months in the glasshouse of the Forestry Research Institute of Oji Holdings Corporation, and irrigated with sufficient amounts of water daily before the drought experiment. To determine drought tolerance, drought and recovery experiments were successively performed. For the drought experiment, Eucalyptus trees were grown for 4 months under low-, middle-, and high-drought levels. The “low-drought level” was determined as the minimum irrigation level that no leaf shedding occurs, which was irrigation of 870 mL of water per each pot every other day. For the middle- and high-drought levels, irrigation of 870 mL of water was reduced every two days and every four days, respectively. Thereafter, irrigation with a sufficient amount of water was started again every two days (recovery experiment). Tree heights, diameters at the base of the stems, and number of leaves were measured monthly during the drought experiment, and then, tree height and shoot and stem fresh weight were measured after the recovery experiment. The duration of the drought and recovery experiment was determined as simulated dry and wet periods in the commercial plantation site for Eucalyptus in Brazil.

Statistical analysis

Differences between the Eucalyptus transgenic lines (EcHB1-2 and EcHB1-10) and the control line GUT5 were tested using ANOVA with Dunnett’s post hoc test. The differences in the traits between GUT5 and the pooled data for the two transgenic lines EcHB1-2 and EcHB1-10 were tested using Welch’s t-test. Principal component analyses were performed to analyze 1) patterns of similarities in the photosynthetic traits and some anatomical traits and 2) patterns of similarities in the leaf anatomical traits. These statistical analyses were performed using R software (ver. 3.2.4)45.