Journal of Experimental Botany

Lawson, Tracy; Leakey, Andrew D B

doi: 10.1093/jxb/erae425pmid: 39545386

Tan, Grace D; Chaudhuri, Ushasi; Varela, Sebastian; Ahuja, Narendra; Leakey, Andrew D B

doi: 10.1093/jxb/erae395pmid: 39363775

Artificial intelligence and machine learning (AI/ML) can be used to automatically analyze large image datasets. One valuable application of this approach is estimation of plant trait data contained within images. Here we review 39 papers that describe the development and/or application of such models for estimation of stomatal traits from epidermal micrographs. In doing so, we hope to provide plant biologists with a foundational understanding of AI/ML and summarize the current capabilities and limitations of published tools. While most models show human-level performance for stomatal density (SD) quantification at superhuman speed, they are often likely to be limited in how broadly they can be applied across phenotypic diversity associated with genetic, environmental, or developmental variation. Other models can make predictions across greater phenotypic diversity and/or additional stomatal/epidermal traits, but require significantly greater time investment to generate ground-truth data. We discuss the challenges and opportunities presented by AI/ML-enabled computer vision analysis, and make recommendations for future work to advance accelerated stomatal phenotyping.

Gibbs, Jonathon A; Burgess, Alexandra J

doi: 10.1093/jxb/erae207pmid: 38716775

Plant physiology and metabolism rely on the function of stomata, structures on the surface of above-ground organs that facilitate the exchange of gases with the atmosphere. The morphology of the guard cells and corresponding pore that make up the stomata, as well as the density (number per unit area), are critical in determining overall gas exchange capacity. These characteristics can be quantified visually from images captured using microscopy, traditionally relying on time-consuming manual analysis. However, deep learning (DL) models provide a promising route to increase the throughput and accuracy of plant phenotyping tasks, including stomatal analysis. Here we review the published literature on the application of DL for stomatal analysis. We discuss the variation in pipelines used, from data acquisition, pre-processing, DL architecture, and output evaluation to post-processing. We introduce the most common network structures, the plant species that have been studied, and the measurements that have been performed. Through this review, we hope to promote the use of DL methods for plant phenotyping tasks and highlight future requirements to optimize uptake, predominantly focusing on the sharing of datasets and generalization of models as well as the caveats associated with utilizing image data to infer physiological function.

Hasan, Md Mahadi; Liu, Xu-Dong; Yao, Guang-Qian; Liu, Jianquan; Fang, Xiang-Wen

doi: 10.1093/jxb/erae060pmid: 38367013

Ethylene, a plant hormone that significantly influences both plant growth and response to stress, plays a well-established role in stress signaling. However, its impact on stomatal opening and closure during dehydration and rehydration remains relatively unexplored and is still debated. Exogenous ethylene has been proven to induce stomatal closure through a series of signaling pathways, including the accumulation of reactive oxygen species, subsequent synthesis of nitric oxide and hydrogen sulfide, and SLOW ANION CHANNEL-ASSOCIATED 1 activation. Thus, it has been suggested that ethylene might function to induce stomatal closure synergistically with abscisic acid (ABA). Furthermore, it has also been shown that increased ethylene can inhibit ABA- and jasmonic acid-induced stomatal closure, thus hindering drought-induced closure during dehydration. Simultaneously, other stresses, such as chilling, ozone pollution, and K+ deficiency, inhibit drought- and ABA-induced stomatal closure in an ethylene synthesis-dependent manner. However, ethylene has been shown to take on an opposing role during rehydration, preventing stomatal opening in the absence of ABA through its own signaling pathway. These findings offer novel insights into the function of ethylene in stomatal regulation during dehydration and rehydration, giving a better understanding of the mechanisms underlying ethylene-induced stomatal movement in seed plants.

Faralli, Michele; Mellers, Greg; Wall, Shellie; Vialet-Chabrand, Silvere; Forget, Guillaume; Galle, Alexander; Van Rie, Jeron; Gardner, Keith A; Ober, Eric S; Cockram, James; Lawson, Tracy

doi: 10.1093/jxb/erae233pmid: 38795361

Recent research has shown that optimizing photosynthetic and stomatal traits holds promise for improved crop performance. However, standard phenotyping tools such as gas exchange systems have limited throughput. In this work, a novel approach based on a bespoke gas exchange chamber allowing combined measurement of the quantum yield of PSII (Fq'/Fm'), with an estimation of stomatal conductance via thermal imaging was used to phenotype a range of bread wheat (Triticum aestivum L.) genotypes. Using the dual-imaging methods and traditional approaches, we found broad and significant variation in key traits, including photosynthetic CO2 uptake at saturating light and ambient CO2 concentration (Asat), photosynthetic CO2 uptake at saturating light and elevated CO2 concentration (Amax), the maximum velocity of Rubisco for carboxylation (Vcmax), time for stomatal opening (Ki), and leaf evaporative cooling. Anatomical analysis revealed significant variation in flag leaf adaxial stomatal density. Associations between traits highlighted significant relationships between leaf evaporative cooling, leaf stomatal conductance, and Fq'/Fm', highlighting the importance of stomatal conductance and stomatal rapidity in maintaining optimal leaf temperature for photosynthesis in wheat. Additionally, gsmin and gsmax were positively associated, indicating that potential combinations of preferable traits (i.e. inherently high gsmax, low Ki, and maintained leaf evaporative cooling) are present in wheat. This work highlights the effectiveness of thermal imaging in screening dynamic gs in a panel of wheat genotypes. The wide phenotypic variation observed suggested the presence of exploitable genetic variability in bread wheat for dynamic stomatal conductance traits and photosynthetic capacity for targeted optimization within future breeding programmes.

Crawford, Joseph D; Twohey, Robert J; Pathare, Varsha S; Studer, Anthony J; Cousins, Asaph B

doi: 10.1093/jxb/erae286pmid: 38970337

The ratio of net CO2 uptake (Anet) and stomatal conductance (gs) is an intrinsic measurement of leaf water use efficiency (WUEi); however, its measurement can be challenging for large phenotypic screens. Measurements of the leaf carbon isotope composition (δ13Cleaf) may be a scalable tool to approximate WUEi for screening because it in part reflects the competing influences of Anet and gs on the CO2 partial pressure (pCO2) inside the leaf over time. However, in C4 photosynthesis, the CO2-concentrating mechanism complicates the relationship between δ13Cleaf and WUEi. Despite this complicated relationship, several studies have shown genetic variation in δ13Cleaf across C4 plants. Yet there has not been a clear demonstration of if Anet or gs are the causal mechanisms controlling WUEi and δ13Cleaf. Our approach was to characterize leaf photosynthetic traits of two Zea mays recombinant inbred lines (Z007E0067 and Z007E0150) which consistently differ for δ13Cleaf even though they have minimal confounding genetic differences. We demonstrate that these two genotypes contrasted in WUEi driven by differences in the speed of stomatal responses to changes in pCO2 and light that lead to unproductive leaf water loss. These findings provide support that differences in δ13Cleaf in closely related genotypes do reflect greater WUEi and further suggest that differences in stomatal kinetic response to changing environmental conditions is a key target to improve WUEi.

Yoshiyama, Yugo; Wakabayashi, Yu; Mercer, Kristin L; Kawabata, Saneyuki; Kobayashi, Takayuki; Tabuchi, Toshihito; Yamori, Wataru

doi: 10.1093/jxb/erae082pmid: 38606772

Plants grown under field conditions experience fluctuating light. Understanding the natural genetic variations for a similarly dynamic photosynthetic response among untapped germplasm resources, as well as the underlying mechanisms, may offer breeding strategies to improve production using molecular approaches. Here, we measured gas exchange under fluctuating light, along with stomatal density and size, in eight wild tomato species and two tomato cultivars. The photosynthetic induction response showed significant diversity, with some wild species having faster induction rates than the two cultivars. Species with faster photosynthetic induction rates had higher daily integrated photosynthesis, but lower average water use efficiency because of high stomatal conductance under natural fluctuating light. The variation in photosynthetic induction was closely associated with the speed of stomatal responses, highlighting its critical role in maximizing photosynthesis under fluctuating light conditions. Moreover, stomatal size was negatively correlated with stomatal density within a species, and plants with smaller stomata at a higher density had a quicker photosynthetic response than those with larger stomata at lower density. Our findings show that the response of stomatal conductance plays a pivotal role in photosynthetic induction, with smaller stomata at higher density proving advantageous for photosynthesis under fluctuating light in tomato species. The interspecific variation in the rate of stomatal responses could offer an untapped resource for optimizing dynamic photosynthetic responses under field conditions.

Al-Salman, Yazen; Cano, Francisco Javier; Mace, Emma; Jordan, David; Groszmann, Michael; Ghannoum, Oula

doi: 10.1093/jxb/erae418pmid: 39377267

Environmental change requires more crop production per water use to meet the rising global food demands. However, improving crop intrinsic water use efficiency (iWUE) usually comes at the expense of carbon assimilation. Sorghum is a key crop in many vulnerable agricultural systems with higher tolerance to water stress (WS) than most widely planted crops. To investigate physiological controls on iWUE and its inheritance in sorghum, we screened 89 genotypes selected based on inherited haplotypes from an elite line or five exotics lines, containing a mix of geographical origins and dry versus milder climates, which included different aquaporin (AQP) alleles. We found significant variation among key highly heritable gas exchange and hydraulic traits, with some being significantly affected by variation in haplotypes among parental lines. Plants with a higher proportion of the non-stomatal component of iWUE still maintained iWUE under WS by maintaining photosynthetic capacity, independently of reduction in leaf hydraulic conductance. Haplotypes associated with two AQPs (SbPIP1.1 and SbTIP3.2) influenced iWUE and related traits. These findings expand the range of traits that bridge the trade-off between iWUE and productivity in C4 crops, and provide possible genetic regions that can be targeted for breeding.

Battle, Martin W; Vialet-Chabrand, Silvere; Kasznicki, Piotr; Simkin, Andrew J; Lawson, Tracy

doi: 10.1093/jxb/erae389pmid: 39292501

In this study, we assessed 43 accessions of sorghum from 16 countries across three continents. Our objective was to identify stomatal and photosynthetic traits that could be exploited in breeding programmes to increase photosynthesis without increasing water use under dynamic light environments. Under field conditions, sorghum crops often have limited water availability and are exposed to rapidly fluctuating light intensities, which influences both photosynthesis and stomatal behaviour. Our results highlight a tight coupling between photosynthetic rate (A) and stomatal conductance (gs) even under dynamic light conditions that results in steady intrinsic water use efficiency (Wi). This was mainly due to rapid stomatal responses, with the majority of sorghum accessions responding within ≤5 min. The maintenance of the ratio of the concentration of CO2 inside the leaf (Ci) and the surrounding atmospheric concentration (Ca) over a large range of accessions suggests high stomatal sensitivity to changes in Ci, that could underlie the rapid gs responses and extremely close relationship between A and gs under both dynamic and steady-state conditions. Therefore, sorghum represents a prime candidate for uncovering the elusive mechanisms that coordinate A and gs, and such information could be used to design crops with high A without incurring significant water losses and eroding Wi.

Taylor, Georgia; Walter, Julia; Kromdijk, Johannes

doi: 10.1093/jxb/erae093pmid: 38442206

The stomatal response to red light appears to link stomatal conductance (gs) with photosynthetic rates. Initially, it was suggested that changes in intercellular CO2 concentration (Ci) provide the main cue via a Ci-dependent response. However, evidence for Ci-independent mechanisms suggests an additional, more direct relationship with photosynthesis. While both Ci-dependent and -independent mechanisms clearly function in stomatal red light responses, little is known regarding their relative contribution. The present study aimed to quantify the relative magnitude of Ci-dependent and -independent mechanisms on the stomatal red light response, to characterize their interplay and to assess the putative link between plastoquinone redox state and Ci-independent stomatal responses. Red light response curves measured at a range of Ci values for wild-type Arabidopsis (Col-0) and the CO2 hyposensitive mutant ca1ca4 allowed deconvolution of Ci-dependent and -independent pathways. Surprisingly, we observed that both mechanisms contribute equally to stomatal red light responses, but Ci-independent stomatal opening is suppressed at high Ci. The present data are also consistent with the involvement of the plastoquinone redox state in coordinating the Ci-independent component. Overall, it seems that while Ci-independent mechanisms are distinct from responses to Ci, interplay between these two pathways is important to facilitate effective coordination between gs and photosynthesis.