Drought is one of the most important environmental factors limiting plant production worldwide and photosynthesis is one of the key targets of water stress, hence the importance of understanding the responses of photosynthesis to drought, and the large number of researchers who are involved in this field of research. I believe this is one of the reasons why the paper is highly cited.

“...the paper ended up in a sort of a crossroad of research interests, being cited by people involved in research on photosynthesis responses to drought, regulation of mesophyll conductance, grapevine research, etc.”

Another factor to consider was the opportune appearance of this paper at a time (2002) when a strong controversy regarding stomatal versus metabolic limitations to photosynthesis under drought was being studied by important research groups such as those of David Lawlor (Rothamsted, UK) and Garbriel Cornic (Orsay, France).

At the same time, a metabolic basis for a finite and variable mesophyll conductance to CO₂ (g_m) was becoming evident following the combined work by groups such as those of Steve Long (Urbana, USA) and Suzanne von Caemmerer (ANU, Australia) showing the temperature dependency of g_m, as well as the work done by Ichiro Terashima’s group (Osaka, Japan), which suggested a role for aquaporins in µm regulation.

In summary, our paper ended up at a kind of a crossroads of research interests, being cited by people involved in research on photosynthesis responses to drought, as well as those studying the regulation of mesophyll conductance, grapevine research, etc.

To be honest, each of the mechanisms described (stomatal closure, regulation of g_m, metabolic impairment) had already been shown separately in the past. The novelty is that we put together all these mechanisms, and determined their importance under progressive drought in field-grown plants. The paper essentially describes, for the first time, a pattern of photosynthesis responses to gradually induced water stress that is now widely accepted to occur in C₃ plants.

Basically, an early step consists in partial stomatal closure, leading to somewhat reduced photosynthesis. As drought intensifies, g_m is down-regulated leading to further reductions in CO₂ availability inside chloroplasts and, thus, photosynthesis. Only when drought is very severe and photosynthesis has almost stopped, a metabolic impairment of photosynthetic components does indeed seem to occur.

First, we were able to show that using stomatal closure as an integrative indicator for the degree of water stress, unified the progressive photosynthetic responses to stress among many different cultivars. Later on, we extended the finding to unify responses among different species. Previous research had mainly focused on leaf-relative water content or water potential as indicators of water stress, leading to different responses depending on the cultivars and/or species used, and hence contributing to controversy about the mechanisms of response.

Second, we showed that the leaf internal diffusion capacity (g_m) changed very dynamically under water stress. While this had already been suggested in the past by many researchers, the most common view at that time was that g_m was essentially related to leaf structure and hence should be mostly constant in response to short-term environmental changes.

Finally, combining both findings, we showed that the previous controversy could be solved, at least in part. Essentially, evidence for early drought-induced photosynthetic metabolism impairment came from the in vivo analysis of photosynthesis responses to CO₂, using gas exchange techniques, which implicitly assumed that g_m was infinite.

By contrast, evidence for stomata-mediated photosynthesis decline came from measurements of stomatal conductance, and from the fact that in vitro analysis of the activity of photosynthetic enzymes usually revealed little or no effect of water stress. By considering a finite and variable g_m during drought, and including this effect in the in vivo analysis, we showed that it revealed a pattern similar to that of an in vitro analysis, i.e., showing little effect on photosynthetic metabolism until drought was severe.

This work was part of my Ph.D. work on grapevine responses to water stress. The group led by my supervisor, Hipólito Medrano, had been involved in both the study of photosynthesis and water stress research for many years. The interest in analyzing these elements in grapevines, came from the fact that irrigation had only been recently allowed for this crop in Spain, leading to increased production but, in most cases, reduced grape and wine quality.

The problems we encountered at that time were related to the strong controversy mentioned above, between researchers defending their belief that stomatal closure was the only cause for decreased photosynthesis under drought, and researchers strongly arguing in favor of an important metabolic impairment, along with the widely extended belief that g_m was only related to leaf structure and hence must be constant during drought stress.

These factors imply that it took quite awhile for people to begin to believe in our results, and the paper was actually rejected by two journals prior to being accepted for publication in Functional Plant Biology. Their editor, Jennifer Henry, strongly favored publication of the manuscript, despite some concerns by the journal’s reviewers.

Mesophyll conductance to CO₂ has been shown to be an important player in photosynthesis regulation, as much as it was previously known for stomatal conductance and Calvin cycle components. Therefore, ongoing research in this field is needed, particularly in order to understand the metabolic components of g_m regulation, as well as to discover, in more detail, to which environmental variables does it respond.

Understanding that drought leads to decreased photosynthesis, not only by decreasing the degree of stomatal opening, but also by decreasing mesophyll conductance or leaf internal CO₂ diffusion, permits the envisaging of a way to improve plant production and water use efficiency for the future.

If we were able to design plants with a strong stomatal response to drought, but with reduced mesophyll response, this would lead to plants with higher photosynthesis to water loss ratios under drought, i.e., plants with improved water-use efficiency. A necessary prior step in achieving this goal would be to clearly identify the molecular basis of mesophyll conductance regulation.