Paul E. Verslues

Proline Metabolism and Its Implications for Plant-Environment Interaction

Proline has long been known to accumulate in plants experiencing water limitation and this has driven studies of proline as a beneficial solute allowing plants to increase cellular osmolarity during water limitation. Proline metabolism also has roles in redox buffering and energy transfer and is involved in plant pathogen interaction and programmed cell death. Some of these unique roles of proline depend on the properties of proline itself, whereas others depend on the “proline cycle” of coordinated proline synthesis in the chloroplast and cytoplasm with proline catabolism in the mitochondria. The regulatory mechanisms controlling proline metabolism, intercellular and intracellular transport and connections of proline to other metabolic pathways are all important to the in vivo functions of proline metabolism. Connections of proline metabolism to the oxidative pentose phosphate pathway and glutamate-glutamine metabolism are of particular interest. The N-acetyl glutamate pathway can also produce ornithine and, potentially, proline but its role and activity are unclear. Use of model systems such as Arabidopsis thaliana to better understand both these long studied and newly emerging functions of proline can help in the design of next-generation experiments testing whether proline metabolism is a promising metabolic engineering target for improving stress resistance of economically important plants.

Essential role of tissue specific proline synthesis and catabolism in growth and redox balance at low water potential

To better define the still unclear role of proline (Pro) metabolism in drought resistance, we analyzed Arabidopsis (Arabidopsis thaliana) Δ1-pyrroline-5-carboxylate synthetase1 (p5cs1) mutants deficient in stress-induced Pro synthesis as well as proline dehydrogenase (pdh1) mutants blocked in Pro catabolism and found that both Pro synthesis and catabolism were required for optimal growth at low water potential (ψw). The abscisic acid (ABA)-deficient mutant aba2-1 had similar reduction in root elongation as p5cs1 and p5cs1/aba2-1 double mutants. However, the reduced growth of aba2-1 but not p5cs1/aba2-1 could be complemented by exogenous ABA, indicating that Pro metabolism was required for ABA-mediated growth protection at low ψw. PDH1 maintained high expression in the root apex and shoot meristem at low ψw rather than being repressed, as in the bulk of the shoot tissue. This, plus a reduced oxygen consumption and buildup of Pro in the root apex of pdh1-2, indicated that active Pro catabolism was needed to sustain growth at low ψw. Conversely, P5CS1 expression was most highly induced in shoot tissue. Both p5cs1-4 and pdh1-2 had a more reduced NADP/NADPH ratio than the wild type at low ψw. These results indicate a new model of Pro metabolism at low ψw whereby Pro synthesis in the photosynthetic tissue regenerates NADP while Pro catabolism in meristematic and expanding cells is needed to sustain growth. Tissue-specific differences in Pro metabolism and function in maintaining a favorable NADP/NADPH ratio are relevant to understanding metabolic adaptations to drought and efforts to enhance drought resistance.

Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo

Osmotic stress associated with drought or salinity is a major factor that limits plant productivity. Protein kinases in the SNF1-related protein kinase 2 (SnRK2) family are activated by osmotic stress, suggesting that the kinases are involved in osmotic stress signaling. However, due to functional redundancy, their contribution to osmotic stress responses remained unclear. In this report, we constructed an Arabidopsis line carrying mutations in all 10 members of the SnRK2 family. The decuple mutant snrk2.1/2/3/4/5/6/7/8/9/10 grew poorly under hyperosmotic stress conditions but was similar to the wild type in culture media in the absence of osmotic stress. The mutant was also defective in gene regulation and the accumulation of abscisic acid (ABA), proline, and inositol 1,4,5-trisphosphate under osmotic stress. In addition, analysis of mutants defective in the ABA-activated SnRK2s (snrk2.2/3/6) and mutants defective in the rest of the SnRK2s (snrk2.1/4/5/7/8/9/10) revealed that SnRK2s are a merging point of ABA-dependent and -independent pathways for osmotic stress responses. These results demonstrate critical functions of the SnRK2s in mediating osmotic stress signaling and tolerance.

Mechanisms independent of abscisic acid (ABA) or proline feedback have a predominant role in transcriptional regulation of proline metabolism during low water potential and stress recovery

Proline accumulation in response to abiotic stress is controlled partially by transcriptional regulation of key enzymes including Δ¹-pyrroline-carboxylate synthetase1 (P5CS1), proline dehydrogenase (ProDH), ornithine amino transferase (OAT) and Δ¹-pyrroline-carboxylate dehydrogenase (P5CDH). For these genes, the role of abscisic acid (ABA), role of feedback regulation by high proline and the mechanisms of gene regulation upon stress release remain unclear. An ABA-deficient (aba2-1) mutant, mutants deficient in proline accumulation (p5cs1), as well as double mutants deficient in both, were used to determine the importance of these factors in transcriptional regulation of proline metabolism. Upregulation of P5CS1 by low water potential was less dependent on ABA than that of stress-marker genes used for comparison. ProDH downregulation by low water potential and upregulation by stress release was not impaired in aba2-1, p5cs1 or p5cs1/aba2-1 compared with wild type despite differing ABA and proline levels in these mutants. Thus, ProDH is a model for characterization of novel regulatory mechanisms associated with low water potential and stress recovery. Both OAT and P5CDH were upregulated during low water potential. This contrasts with previous salt stress experiments and raises questions about the flux of metabolites through proline metabolism under low water potential when high levels of proline accumulate.

Unique Drought Resistance Functions of the Highly ABA-Induced Clade A Protein Phosphatase 2Cs

Six Arabidopsis (Arabidopsis thaliana) clade A protein phosphatase 2Cs (PP2Cs) have established abscisic acid (ABA) signaling roles; however, phenotypic roles of the remaining three “HAI” PP2Cs, Highly ABA-Induced1 (HAI1), AKT1-Interacting PP2C1/HAI2, and HAI3, have remained unclear. HAI PP2C mutants had enhanced proline and osmoregulatory solute accumulation at low water potential, while mutants of other clade A PP2Cs had no or lesser effect on these drought resistance traits. hai1-2 also had increased expression of abiotic stress-associated genes, including dehydrins and late embryogenesis abundant proteins, but decreased expression of several defense-related genes. Conversely, the HAI PP2Cs had relatively less impact on several ABA sensitivity phenotypes. HAI PP2C single mutants were unaffected in ABA sensitivity, while double and triple mutants were moderately hypersensitive in postgermination ABA response but ABA insensitive in germination. The HAI PP2Cs interacted most strongly with PYL5 and PYL7 to -10 of the PYL/RCAR ABA receptor family, with PYL7 to -10 interactions being relatively little affected by ABA in yeast two-hybrid assays. HAI1 had especially limited PYL interaction. Reduced expression of the main HAI1-interacting PYLs at low water potential when HAI1 expression was strongly induced also suggests limited PYL regulation and a role of HAI1 activity in negatively regulating specific drought resistance phenotypes. Overall, the HAI PP2Cs had greatest effect on ABA-independent low water potential phenotypes and lesser effect on classical ABA sensitivity phenotypes. Both this and their distinct PYL interaction demonstrate a new level of functional differentiation among the clade A PP2Cs and a point of cross talk between ABA-dependent and ABA-independent drought-associated signaling.

Drought, metabolites, and Arabidopsis natural variation: a promising combination for understanding adaptation to water-limited environments

Drought elicits substantial changes in plant metabolism and it remains a challenge to determine which of these changes represent adaptive responses and which of them are merely neutral effects or even symptoms of damage. Arabidopsis primarily uses low water potential/dehydration avoidance strategies to respond to water limitation. The large variation in evolved stress responses among accessions can be a powerful tool to identify ecologically important and adaptive traits; however, collection of relevant phenotype data under controlled water stress is often a limiting factor. Quantitative genetics of Arabidopsis has great potential to find the genes underlying variation in drought-affected metabolic traits, for example proline metabolism, as well as overall adaptation.

Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation.

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ1-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation.

LWR1 and LWR2 Are Required for Osmoregulation and Osmotic Adjustment in Arabidopsis

With the goal of identifying molecular components of the low-water-potential response, we have carried out a two-part selection and screening strategy to identify new Arabidopsis mutants. Using a system of polyethylene glycol-infused agar plates to impose a constant low-water-potential stress, putative mutants impaired in low-water-potential induction of the tomato (Lycopersicon esculentum) le25 promoter were selected. These lines were then screened for altered accumulation of free Pro. The seedlings of 22 mutant lines had either higher or lower Pro content than wild type when exposed to low water potential. Two mutants, designated low-water-potential response1 (lwr1) and lwr2, were characterized in detail. In addition to higher Pro accumulation, lwr1 seedlings had higher total solute content, greater osmotic adjustment at low water potential, altered abscisic acid content, and increased sensitivity to applied abscisic acid with respect to Pro content. lwr1 also had altered growth and morphology. lwr2, in contrast, had lower Pro content and less osmotic adjustment leading to greater water loss at low water potential. Both lwr1 and lwr2 also had altered leaf solute content and water relations in unstressed soil-grown plants. In both mutants, the effects on solute content were too large to be explained by the changes in Pro content alone, indicating that LWR1 and LWR2 affect multiple aspects of cellular osmoregulation.

Genome-Wide Association Mapping Combined with Reverse Genetics Identifies New Effectors of Low Water Potential-Induced Proline Accumulation in Arabidopsis

Genome-wide association analysis and spatial patterning of single-nucleotide polymorphism data guide reverse genetics to identify new genes linking stress-induced proline accumulation with cellular redox and energy status. Arabidopsis (Arabidopsis thaliana) exhibits natural genetic variation in drought response, including varying levels of proline (Pro) accumulation under low water potential. As Pro accumulation is potentially important for stress tolerance and cellular redox control, we conducted a genome-wide association (GWAS) study of low water potential-induced Pro accumulation using a panel of natural accessions and publicly available single-nucleotide polymorphism (SNP) data sets. Candidate genomic regions were prioritized for subsequent study using metrics considering both the strength and spatial clustering of the association signal. These analyses found many candidate regions likely containing gene(s) influencing Pro accumulation. Reverse genetic analysis of several candidates identified new Pro effector genes, including thioredoxins and several genes encoding Universal Stress Protein A domain proteins. These new Pro effector genes further link Pro accumulation to cellular redox and energy status. Additional new Pro effector genes found include the mitochondrial protease LON1, ribosomal protein RPL24A, protein phosphatase 2A subunit A3, a MADS box protein, and a nucleoside triphosphate hydrolase. Several of these new Pro effector genes were from regions with multiple SNPs, each having moderate association with Pro accumulation. This pattern supports the use of summary approaches that incorporate clusters of SNP associations in addition to consideration of individual SNP probability values. Further GWAS-guided reverse genetics promises to find additional effectors of Pro accumulation. The combination of GWAS and reverse genetics to efficiently identify new effector genes may be especially applicable for traits difficult to analyze by other genetic screening methods.

Role of the Putative Osmosensor Arabidopsis Histidine Kinase1 in Dehydration Avoidance and Low-Water-Potential Response

Summary: AHK1 mutants had increased stomatal density and faster leaf water loss; however, AHK1 mutants were not more sensitive to controlled low water potential stress and not impaired in abscisic acid, Pro, or osmoregulatory solute accumulation, thus indicating that AHK1 is not the main plant osmosensor controlling these drought tolerance-associated phenotypes. The molecular basis of plant osmosensing remains unknown. Arabidopsis (Arabidopsis thaliana) Histidine Kinase1 (AHK1) can complement the osmosensitivity of yeast (Saccharomyces cerevisiae) osmosensor mutants lacking Synthetic Lethal of N-end rule1 and SH3-containing Osmosensor and has been proposed to act as a plant osmosensor. We found that ahk1 mutants in either the Arabidopsis Nossen-0 or Columbia-0 background had increased stomatal density and stomatal index consistent with greater transpirational water loss. However, the growth of ahk1 mutants was not more sensitive to controlled moderate low water potential (ψw) or to salt stress. Also, ahk1 mutants had increased, rather than reduced, solute accumulation across a range of low ψw severities. ahk1 mutants had reduced low ψw induction of Δ1-Pyrroline-5-Carboxylate Synthetase1 (P5CS1) and 9-cis-Epoxycarotenoid Dioxygenase3, which encode rate-limiting enzymes in proline and abscisic acid (ABA) synthesis, respectively. However, neither Pro nor ABA accumulation was reduced in ahk1 mutants at low ψw. P5CS1 protein level was not reduced in ahk1 mutants. This indicated that proline accumulation was regulated in part by posttranscriptional control of P5CS1 that was not affected by AHK1. Expression of AHK1 itself was reduced by low ψw, in contrast to previous reports. These results define a role of AHK1 in controlling stomatal density and the transcription of stress-responsive genes. These phenotypes may be mediated in part by reduced ABA sensitivity. More rapid transpiration and water depletion can also explain the previously reported sensitivity of ahk1 to uncontrolled soil drying. The unimpaired growth, ABA, proline, and solute accumulation of ahk1 mutants at low ψw suggest that AHK1 may not be the main plant osmosensor required for low ψw tolerance.