**3. Internal signals**

#### **3.1 Ion channels and transmembrane antiporters**

There is no question that stomatal movements (stomatal opening and closing) of seed plants, including crop plants, arise from the transport, accumulation, and release of osmotically active solutes (reviewed by [32]). It has been shown that the guard cell movement is controlled by movement of Cl+ , Na+ , K+ , and also changes in the sucrose and malate levels [32, 33]. It is reasonable to give expectation that ion exchange, inducing change in pH, might indirectly determine response time of stomatal adjustments during light fluctuations based on previous literatures. For example, membrane depolarization in ABA stimulates K+ efflux within seconds through outward-rectifying K+ channels, in Arabidopsis the GORK K+ channel [34, 35], and these K+ currents are enhanced during the subsequent 3–5 min as a consequence of rise in cytosolic pH [36, 37]. Stomatal aperture responds more slowly, typically with half-times of 10– 20 min, reaching a new stable, (near) closed state after 45–60 min [38–40]. Thus, making a connection of ion channel antiporters to the speed and efficacy of stomatal movements is necessarily important.

#### **3.2 Anatomical features of stomata**

Responsiveness of stomatal adjustments under changing environments is also dependent on anatomical characteristics. In fact, stomatal anatomical features define the maximum theoret‐ ical conductance and also influence the speed of response [41]. Many experimental evidences have demonstrated that stomatal density is negatively correlated with stomatal size [42, 43]. The interaction/correlation between stomatal size and density and the impact on stomatal function have received much attention [44]. The latest studies have also implied that physical attributes affect stomatal response times following environmental perturbations [45]. There‐ fore, it is possible to manipulate the stomatal structure, for example, we can take into consid‐ eration the interaction between stomatal size and number and its impact on rapidity of stomatal movement.

#### **3.3 Casual genes of stomatal features**

0.0

**MDH activity (μmol m−2 s−1)**

7.9 ± 3.4 (24.8 ± 3.3)

14.9 ± 1.2 (26.5 ± 0.4)

17.2 ± 0.3 (26.1 ± 0.2)

19.0 ± 2.7 (28.9 ± 0.4)

12.1 ± 1.7 (29.0 ± 1.2)

20.9 ± 0.4 (28.8 ± 1.5)

14.6 ± 1.7 (28.6 ± 0.6)

13.7 ± 0.9 (31.6 ± 1.1)

Solar irradiation (µmol m–2s–1)

09:00:00

0

10

20

*PN* (µmol m–2s–1)

data).

30

40

50

10:00:00

Stomatal delays

**OTCs Heat PEPC activity**

aft4 19.7 ± 0.5

aft4 15.8 ± 0.7

aft4 17.9 ± 1.3

aft4 12.8 ± 1.0

B76 aft0 15.5 ± 0.5

80 Applied Photosynthesis - New Progress

B106 aft0 13.3 ± 0.7

B76 aft0 13.6 ± 1.5

B106 aft0 10.5 ± 1.7

*Ambient[CO2]*

*Elevated[CO2]*

**(μmol m−2 s−1)**

(33.1 ± 1.6)

(36.5 ± 1.5)

(32.8 ± 0.5)

(36.6 ± 1.4)

(32.0 ± 0.7)

(34.4 ± 0.9)

(32.2 ± 0.1)

(31.9 ± 1.0)

Values of control experiments were shown in brackets (Qu et al. 2016, unpublished data).

11:00:00

12:00:00

Daily time (h:m:s)

13:00:00

**Figure 3.** Photosynthesis and stomatal conductance in response to naturally light regime (Qu et al. 2016, unpublished

14:00:00

15:00:00

16:00:00

.2

.4

.6

*gs* (mmol m–2s–1)

.8

1.0

*gs PAR*

**ME activity (μmol m−2 s−1)**

3.7 ± 0.4 (31.6 ± 0.5)

20.6 ± 0.3 (29.0 ± 0.1)

12.4 ± 1.1 (24.6 ± 0.3)

13.9 ± 0.7 (27.7 ± 0.3)

10.9 ± 0.2 (25.3 ± 0.4)

18.1 ± 1.0 (26.3 ± 0.6)

9.0 ± 0.6 (26.7 ± 0.8)

11.6 ± 1.6 (29.2 ± 0.5)

% Change 27.1 456.8 88.6

% Change 18.8 12.1 10.5

% Change 31.6 66.5 72.7

% Change 21.9 29.1 -6.2

**Table 1.** Enzyme activities of PEPC, NADP-ME, and NADP-MDH for B76 vs. B106 grown ambient and elevated [CO2].

1.2

<sup>1800</sup> *<sup>A</sup>*

Engineering and breeding crops for enhanced drought resistance become a pressing task for plant biologists and breeders. Manipulation on functional genes underlying dynamics of stomatal responses and steady-state values of *g*s would be helpful for optimizing WUE and drought resistance of plants [46–51]. For example, mutation in the *SLAC1* gene, which codes for an anion channel, causes slow stomatal opening by light, low CO2, and elevated air humidity in intact plants, due to severely reduced activity of inward K+ channels in *slac1* guard cells [52]. Arabidopsis (*Arabidopsis thaliana*) stomatal density and distribution (*sdd1-1*) mutants, having a point mutation in a single gene that encodes a subtilisin-like Ser protease, exhibit a 2.5-fold higher stomatal density compared with their wild type [53]. Stomatal movements can also be stimulated by membrane fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SYP121), Eisenach et al. [54] demonstrated that stomatal opening and the rise in stomatal transpiration of the *syp121* mutant were delayed in the dark– light transition and following the Ca2+-evoked closure. The increase in stomatal density translates leads to an increase in *g*s and 30% greater *P*N under high light conditions [55]. Tanaka et al. [56] have used plants overexpressing STOMAGEN, a positive regulator of stomatal density, to produce transgenic plants with a two- to three fold greater stomatal density than the wild type. *P*<sup>N</sup> in these plants is increased by 30% due to greater CO2 diffusion into the leaf rather than changes in photosynthetic carboxylation capacity [56]. By contrast, some genes can induce low stomatal density and *g*<sup>s</sup> at high light intensities, for example, upregulation of *sdd1* can restrict CO2 diffusion limited *P*N to 80% of the wild type [57].

These findings exemplify the role of both the physical and functional stomatal features in determining *g*s. In particular, these works illustrate the importance of surrounding environ‐ mental conditions and ion exchange on stomatal behavior and the significance of examining *g* s limitation on *P*N at fluctuating light and elevated CO2 and heat stress.
