► Identifying genetic markers for yield requires rapid quantification of crop traits. ► Proximal sensing offers promise for field-based phenotyping (FBP). ► Efficient data integration and ...modeling-assisted analysis are key for FBP. ► FBP scaled to thousands of field plots is a feasible, attainable goal. ► FBP systems require new, integrative collaborations that cross disciplines.
A major challenge for crop research in the 21st century is how to predict crop performance as a function of genetic architecture. Advances in “next generation” DNA sequencing have greatly improved genotyping efficiency and reduced genotyping costs. Methods for characterizing plant traits (phenotypes), however, have much progressed more slowly over the past 30 years, and constraints in phenotyping capability limit our ability to dissect the genetics of quantitative traits, especially those related to harvestable yield and stress tolerance. As a case in point, mapping populations for major crops may consist of 20 or more families, each represented by as many as 200 lines, necessitating field trials with over 20,000 plots at a single location. Investing in the resources and labor needed to quantify even a few agronomic traits for linkage with genetic markers in such massive populations is currently impractical for most breeding programs. Herein, we define key criteria, experimental approaches, equipment and data analysis tools required for robust, high-throughput field-based phenotyping (FBP). The focus is on simultaneous proximal sensing for spectral reflectance, canopy temperature, and plant architecture where a vehicle carrying replicated sets of sensors records data on multiple plots, with the potential to record data throughout the crop life cycle. The potential to assess traits, such as adaptations to water deficits or acute heat stress, several times during a single diurnal cycle is especially valuable for quantifying stress recovery. Simulation modeling and related tools can help estimate physiological traits such as canopy conductance and rooting capacity. Many of the underlying techniques and requisite instruments are available and in use for precision crop management. Further innovations are required to better integrate the functions of multiple instruments and to ensure efficient, robust analysis of the large volumes of data that are anticipated. A complement to the core proximal sensing is high-throughput phenotyping of specific traits such as nutrient status, seed composition, and other biochemical characteristics, as well as underground root architecture. The ability to “ground truth” results with conventional measurements is also necessary. The development of new sensors and imaging systems undoubtedly will continue to improve our ability to phenotype very large experiments or breeding nurseries, with the core FBP abilities achievable through strong interdisciplinary efforts that assemble and adapt existing technologies in novel ways.
Arabidopsis (Arabidopsis thaliana) plants subjected to water deficit, sodium chloride (NaCl), or abscisic acid treatments were shown to exhibit a significant increase in the amount of leaf cuticular ...lipids. These stress treatments led to increases in cuticular wax amount per unit area of 32% to 80%, due primarily to 29% to 98% increases in wax alkanes. Of these treatments, only water deficit increased the total cutin monomer amount (by 65%), whereas both water deficit and NaCl altered the proportional amounts of cutin monomers. Abscisic acid had little effect on cutin composition. Water deficit, but not NaCl, increased leaf cuticle thickness (by 49%). Electron micrographs revealed that both water-deprived and NaCl-treated plants had elevated osmium accumulation in their cuticles. The abundance of cuticle-associated gene transcripts in leaves was altered by all treatments, including those performed in both pot-grown and in vitro conditions. Notably, the abundance of the ECERIFERUM1 gene transcript, predicted to function in alkane synthesis, was highly induced by all treatments, results consistent with the elevated alkane amounts observed in all treatments. Further, this induction of cuticle lipids was associated with reduced cuticle permeability and may be important for plant acclimation to subsequent water-limited conditions. Taken together, these results show that Arabidopsis provides an excellent model system to study the role of the cuticle in plant response to drought and related stresses, and its associated genetic and cellular regulation.
Abstract Wax biosynthesis is closely controlled by many regulators under different environmental conditions. We have previously shown that the module miR156–SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 ...(SPL9)–DEWAX is involved in the diurnal regulation of wax production; however, it was not determined whether other SPLs are also involved in wax synthesis. Here, we report that SPL13 also regulates drought-induced wax production, by directly and indirectly affecting the expression of the two wax biosynthesis genes ECERIFERUM1 (CER1) and CER4, respectively. In addition, we show that SPL13 together with SPL9 redundantly regulates wax accumulation under both normal and drought stress conditions, and that simultaneous mutation of both genes additively increases cuticle permeability and decreases drought tolerance. However, in contrast to SPL9, SPL13 does not seem to participate in the DEWAX-mediated diurnal regulation of wax production.
Water deficit stress negatively affects wheat growth, physiology, and yield. In lab and hydroponic experiments, osmotic stress levels (control, −2, −4, −6 and −8 Bars) created by PEG-6000, caused a ...significant decline in germination, mean germination time, root, shoot, and coleoptile length in both wheat genotypes examined. Germination was inhibited more in Wafaq-2001 than in Chakwal-50. Wafaq-2001 showed a higher susceptibility index based on root and shoot dry weight than did Chakwal-50. Wheat plants exhibited osmotic adjustment through the accumulation of proline, soluble sugars, soluble proteins, and free amino acids, and increased antioxidation activities of superoxide dismutase, peroxidase, catalase, and malondialdehyde. Increasing water deficit stress caused a linear decline in chlorophyll contents, leaf membrane stability, and relative water content in all wheat plants, with Wafaq-2001 showing a more severe negative impact on these parameters with increasing stress levels. The results suggest the possibility of utilizing some of these parameters as quantitative indicators of water stress tolerance in plants. Gas exchange measurements (photosynthesis, transpiration, stomatal conductance), leaf osmotic potential, water potential, and yield attributes decreased more abruptly with increasing water deficit, whereas leaf cuticular wax content increased in both genotypes, with more severe impacts on Wagaq-2001. More reduction in biochemical, physiological, and yield attributes was observed in Wafaq-2001 than was observed in Chakwal-50. Based on these results, we can conclude that Chakwal-50 is a more drought-tolerant genotype, and has excellent potential for future use in breeding programs to improve wheat drought tolerance.
Abstract Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and ...dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA 3 ) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana . Herewith, the media supplemented with 2 mg/L and 4 mg/L GA 3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA 3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA 3 resulted in the highest accumulation of proline (0.99 μg/mL) as compared to controls (0.37 μg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA 3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA 3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.
Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially ...on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C₂₄) and hexacosanoic acid (C₂₆). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast DoalO (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetasel (lacsl), and lacsl and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.
Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of ...CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀-C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1, and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.
SUMMARY
The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water ...deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound‐ and JA‐responsive gene (CYP96A4) encoding an ER‐localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound‐induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane‐forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.
Significance Statement
Plant cuticle is a lipidic barrier composed of cuticular wax and cutin polymers. Cuticle plays a critical role in protecting plants from various biotic and abiotic stresses. Wounds due to mechanical damage or insect feeding are common and are potentially harmful to plants. However, the effect of wounding on plant cuticular wax and cutin monomer biosynthesis remains unclear. In our study, the findings present the validation that the Arabidopsis cuticle development was positively regulated by wounding mainly through the jasmonic acid (JA) signaling pathway. We also characterized the wound‐ and JA‐responsive gene CYP96A4, which is required for wound‐ and JA‐induced synthesis of cuticular wax and cutin monomer.
Summary
Genetic signature of climate adaptation has been widely recognized across the genome of many organisms; however, the eco‐physiological basis for linking genomic polymorphisms with local ...adaptations remains largely unexplored.
Using a panel of 218 world‐wide Arabidopsis accessions, we characterized the natural variation in root suberization by quantifying 16 suberin monomers. We explored the associations between suberization traits and 126 climate variables. We conducted genome‐wide association analysis and integrated previous genotype–environment association (GEA) to identify the genetic bases underlying suberization variation and their involvements in climate adaptation.
Root suberin content displays extensive variation across Arabidopsis populations and significantly correlates with local moisture gradients and soil characteristics. Specifically, enhanced suberization is associated with drier environments, higher soil cation‐exchange capacity, and lower soil pH; higher proportional levels of very‐long‐chain suberin is negatively correlated with moisture availability, lower soil gravel content, and higher soil silt fraction. We identified 94 putative causal loci and experimentally proved that GPAT6 is involved in C16 suberin biosynthesis. Highly significant associations between the putative genes and environmental variables were observed.
Roots appear highly responsive to environmental heterogeneity via regulation of suberization, especially the suberin composition. The patterns of suberization–environment correlation and the suberin‐related GEA fit the expectations of local adaptation for the polygenic suberization trait.
The plant cuticle, a structure primarily composed of wax and cutin, forms a continuous coating over most aerial plant surfaces. The cuticle plays important roles in plant tolerance to environmental ...stress, including stress imposed by drought. Some members of the 3-KETOACYL-COA SYNTHASE (KCS) family are known to act as metabolic enzymes involved in cuticular wax production. Here we report that Arabidopsis (Arabidopsis thaliana) KCS3, which was previously shown to lack canonical catalytic activity, instead functions as a negative regulator of wax metabolism by reducing the enzymatic activity of KCS6, a key KCS involved in wax production. We demonstrate that the role of KCS3 in regulating KCS6 activity involves physical interactions between specific subunits of the fatty acid elongation complex and is essential for maintaining wax homeostasis. We also show that the role of the KCS3-KCS6 module in regulating wax synthesis is highly conserved across diverse plant taxa from Arabidopsis to the moss Physcomitrium patens, pointing to a critical ancient and basal function of this module in finely regulating wax synthesis.
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