Drawing the future

Abstract

Gas exchange between the plant and the atmosphere is regulated by controlling both the stomatal density and the aperture of the stomatal pore. Environmental factors such as light, the level of atmospheric CO₂ and hormones regulate stomatal development and/or function. Because atmospheric CO₂ levels have been rising since the Industrial Revolution, and it is predicted that they will continue doing so in the future, an understanding of the CO₂ signalling mechanisms in the stomatal responses will help to know how plants were in the past and will allow predicting how they will respond to climate change in the near future. This article covers the recent knowledge of the CO₂ signalling mechanisms that regulate both stomatal function and development.

Introduction

The stomata are epidermal structures formed by a pair of guard cells that function to vary the width of a pore. Opening of the stomatal pore by increasing the turgor status of the guard cells allows CO₂ to diffuse from the atmosphere to inside leaf, but also allows precious water vapour to diffuse out of the leaf.¹ Almost all of the CO₂ fixed by terrestrial plants and most of the water transpired pass though the stomatal pores. The increase of guard-cell turgor is driven by the activation of the guard-cell plasma membrane H⁺-ATPase, creating an inside-negative electrical potential across the membrane, which is required to drive K⁺ uptake in guard cells through inward-rectifying K⁺ channels.² Constitutive activation of this H⁺-ATPase prevents abscisic acid-mediated stomatal closure.³ Plants must regulate the size of their stomatal pores to take in as much CO₂ as possible while losing the least amount of water to prevent their desiccation.¹ However, water loss by transpiration is also an essential process for plant survival, providing the driving force for water and nutrient transport from the roots to the aerial tissues and also preventing leaf warming.⁴

Carbon dioxide is accumulating in the atmosphere much faster than scientists expected, with fossil fuel being the primary source of this increase. Before the Industrial Revolution 200 years ago, atmospheric CO₂ was stable at about 280 ± 10 parts per million (ppm) for several thousand years (Fig. 1).⁵ Current levels of carbon dioxide in the atmosphere reach an average value of 384 ppm, about 100 ppm higher than before the Industrial Revolution (Fig. 1). Therefore, the increase of atmospheric CO₂ is not just an issue of the future: today's plants are growing at an elevated CO₂ concentration that has not been exceeded during the past 420,000 years, and likely not during the past 20 million years.^5–7 Several plausible scenarios help to predict how much the atmospheric CO₂ will increase.⁸ In the most pessimistic outcome, atmospheric CO₂ levels are projected to reach 970 ppm in the year 2100. The most optimistic view anticipates that CO₂ concentrations might stabilize around 540 ppm.

Figure 1.

Past and future CO₂ atmospheric concentrations. Since preindustrial times, the atmospheric concentration of CO₂ has grown significantly. The current CO₂ level (around 384 ppm) is about 100 ppm higher than before the Industrial Revolution. This CO₂ level is the highest for 420,000 years, and probably the highest for the past 20 million years. Several scenarios under contrasting CO₂ emission-control projections have been proposed: in the most optimistic one, CO₂ level might stabilize around 540 ppm; in the most pessimistic outcome, CO₂ level might reach 970 ppm in the year 2100. Modified from the Intergovernmental Panel on Climate Change (IPCC; http://www.ipcc.ch/).

It is well known that, in general, CO₂ concentrations below ambient both stimulate stomatal opening and increase the stomatal density, while CO₂ concentrations above ambient induce the opposite effect.^9,10 The rise in atmospheric CO₂ level is increasing the rate at which CO₂ diffuses into substomatal cavities, and plants are responding by reducing both the number and the size of their stomata to reduce water loss by transpiration while allowing the required level of photosynthesis to be maintained. This might impair the transport of water and nutrients from the soil to the leaves and so increase leaf temperature. This article focuses on the genetic control of the CO₂ signalling mechanisms that regulate both stomatal function and development. The effects of increases in CO₂ concentration on the cell biology have been the subject of much research summarized in detailed reviews (see refs. ⁹ and ¹¹).

Responses of Cuticular Waxes to Increased CO₂

The first evidence that links CO₂ atmospheric levels and stomatal development came from the observation of herbarium specimens collected over the last 200 years. Ian Woodward unravelled that eight arboreal species have experienced a decrease in the stomatal density of their leaves in the last 200 years, which suggested that such decrease was due to the increase of CO₂ concentration during the Industrial Revolution.¹² Laboratory experiments in which plants were grown at preindustrial CO₂ levels confirmed that the decrease of both the stomatal density (number of stomata/area) and the stomatal index ([stomatal density/(stomatal density + epidermal-cell density) × 100], where epidermal cells include guard cells as well as others that constitute the epidermis) was due to an increase over CO₂ preindustrial levels.^12–14 Increased atmospheric current CO₂ level often but not always leads to a decrease in the stomatal index.¹⁴ In some plant species, enrichment of the present atmospheric CO₂ levels induces none or only minor changes in the stomatal density and/or index,^14–19 which suggests that the stomatal response to CO₂ in such as species might be close to the saturation with the current CO₂ level.²⁰ In other species, an increase of the atmospheric CO₂ levels leads to an increase in the stomatal index.^14,18,21 In Arabidopsis accessions, the stomatal density response to CO₂ enrichment broadly parallels interspecific observations.²² Since the atmospheric CO₂ level exerts its action on stomatal development, the number of stomata in fossil plants can be used as a way of tracking CO₂ variation throughout time.^22,24

More than a decade was required to unravel the first genetic evidence underlying the stomatal development response to CO₂ levels. Gray and coworkers found that a putative 3-keto acyl coenzyme A synthase, named HIGH CARBON DIOXIDE (HIC), prevents an increase in the stomatal index in response to atmospheric CO₂ enrichment (1000 ppm ± 100).²⁵ But stomatal functioning appears to be normal.²⁶ An increase in the stomatal index under these conditions may have dramatic consequences for plant physiology as it would increase water loss. Because these enzymes are involved in synthesizing fatty acids with very long chains found in the cuticle,²⁷ it was inferred that in hic mutants an alteration to cuticular composition interferes with the diffusion of an hypothetical inhibitory factor that is stimulated by CO₂ enrichment, increasing the stomatal index (Fig. 2).²⁵ Because hic does not develop stomatal clusters, it is likely that a very limited diffusion of the inhibitory factor might prevent such as structures.²⁰

Figure 2.

Genes associated with CO₂ concentration. Plants must take in as much CO₂ as possible while losing the least amount of water. Under CO₂ concentrations below the current level they develop more stomata with more open pores. Under CO₂ concentrations above the ambient, they exhibit opposite responses. The putative kinase, named HT1 (HIGH LEAF TEMPERATURE1) regulates stomatal function by blocking stomatal closure under lower CO₂ levels that current ones. In contrast, a putative 3-keto-acyl coenzyme A synthase named HIC (HIGH CARBON DIOXIDE) controls stomatal development in response to atmospheric CO₂ enrichment by negatively regulating the formation of an excess of stomata.

Support for the inhibitory factor came from the observation of some Arabidopsis cuticular wax-deficient mutants, which develop abnormal stomatal patterns.^25,28 Both eceriferum-1 and eceriferum-6, which have alteration in wax composition,²⁹ have higher stomatal indices than those of their corresponding wild-type parent.²⁵ By contrast, wax2, which has also alterations in the cuticular wax composition, has a lower stomatal index than the wild type.²⁸ Because these phenotypes were detected at current CO₂ levels, it seems that in general cuticular wax composition is essential to pattern stomata under both high and current CO₂ levels.

But, how is the CO₂ signal detected? Lake and coworkers treated, by using a gas-tight cuvette system, with different atmospheric CO₂ concentrations mature and young leaves of the same individuals.³⁰ The stomatal index and density of developing leaves reflected the CO₂ level experienced by the mature ones. This easy and elegant experiment showed that mature leaves detect atmospheric CO₂ levels and transmit this signal to new ones, which respond to such signal by adjusting their stomatal index and density.³⁰ However, the signal that is believed is transmitted from mature to developing leaves is largely unknown. This systemic response has been also observed in Sinapsis alba,³¹ and it has been proposed that might be a general plant response.³² In addition, mutant analysis strongly suggests that the hormones abscisic acid, ethylene and jasmonates are implicated in this systemic response and that their action might be mediated by reactive oxygen species.³²

Involvement of a Kinase in Response to CO₂ Concentrations Below Current Ambient Levels

Stomatal opening depends on a number of external and internal signals, such as light, CO₂, air humidity and abscisic acid.^2,9,33 The putative kinase HT1 (HIGH LEAF TEMPERATURE1) blocks stomatal closure mainly under lower CO₂ levels than current atmospheric ones (100 ppm), but also under current levels, which allows CO₂ uptake and leaf cooling caused by the transpiration (Fig. 2).³⁴ The stomatal density under these atmospheric conditions is entirely normal,³⁴ suggesting that this gene does not control stomatal development. Stomatal closure under theses conditions might have drastic consequences by blocking CO₂ uptake, and so impairing photosynthesis.³⁵ HT1 is primarily expressed where it exerts its action, in the guard cells,^34,36 although such expression is not regulated by CO₂ concentration.³⁴ Interestingly, ht1 mutants open their pores in response to the fungal toxic fusicoccin,³⁴ an activator of the guard-cell plasma membrane H⁺-ATPase,³⁷ which is telling us that HT1 does not control such H⁺-ATPase nor its downstream targets.³⁵

Although at current CO₂ levels, the stomatal pores of ht1-mutant are less open than those of wild-type plants, they close tighter in response to abscisic acid.³⁴ This suggests that HT1 is not involved in the abscisic acid signalling pathway. However, HT1 regulates stomatal opening in response to blue light. Stomatal conductance increases dramatically in the wild-type plants in response to blue light, but only partially in the ht1 mutants.³⁴ Because blue light regulates stomatal opening by activating the plasma membrane H⁺-ATPase,^38–40 it is likely that HT1 transduces both CO₂ and blue-light signals to the plasma membrane H⁺-ATPase in guard cells inducing stomatal opening.³⁵

Conclusions

Many plants respond to the recent CO₂ increase by reducing both the size of the stomatal aperture and the number of stomata. Like this, they reduce transpiration without inhibiting CO₂ assimilation. HT1 mainly increases stomatal opening under lower CO₂ levels than current -and preindustrial- atmospheric ones.³⁴ However, it does not seem to regulate stomatal function under higher CO₂ level than those of today (700 ppm), as ht1 shows no stomatal defect at such CO₂ concentration.³⁴ It is likely that other genes will regulate stomatal function under future CO₂ concentrations. Although their molecular nature remains unknown, the stomatal response to CO₂ enrichment is not enigmatic. HIC gene regulates stomatal development by preventing an increase in the number of stomata in response to CO₂ enrichment.²⁵ Such excess of stomata could be detrimental to plant survival because of the resulting high water loss and low leaf temperature. Plants have the ability to adapt to challenging environmental conditions by modulating both stomatal function and development so that they take in as much CO₂ as possible while ensuring the least amount of water loss. We now know some of the genes that make it possible. Future challenges to predict the future of the current plants include unravelling HIC-mediated signalling pathway (and its hypothetical interaction with other signalling networks) and uncovering the genes responsible for the stomatal closure under high CO₂ levels.