Reciprocal iron-sulfur regulation in durum wheat converges on a few metabolic hubs that integrate nutrient status with plant growth and performance. Iron and sulfur availabilities were factorially modulated in hydroponically grown wheat to establish how each nutrient and their interaction shape growth, ionome, amino acid and organic acid metabolism, and the expression of key S- and Fe-homeostasis genes. Sulfur availability strongly shaped plant responses to iron availability, determining biomass allocation, ionomic patterns, and the expression of sulfate transporters, sulfur-assimilatory enzymes, and iron homeostasis genes. Metabolomics highlighted citrate, O-acetylserine, and methionine as root metabolites most consistently associated with combined Fe × S treatments. Correlation analysis linked these variables with markers of sulfate transport and assimilation, defining a reproducible interaction signature across biological layers. Sulfur deficiency, especially when combined with low iron, also increased free asparagine accumulation in vegetative tissues, indicating a strong reallocation in nitrogen metabolism under dual stress. Sulfur availability is a major modulator of iron-responsive phenotypic, transcriptional, and metabolic traits in durum wheat and provides a systems-level framework for future functional studies of Fe/S crosstalk.

Sulfate deprivation (-S) results in numerous metabolic and phenotypic alterations in plants. Kinases are often key players in transducing nutrient status signals to molecular components involved in metabolic and developmental program regulation, but despite the physiological importance of sulfur, to date, no signaling kinases have been identified in sulfur-deficiency signaling response programs. Here, we show that the serine/threonine protein kinase CIPK14/SNRK3.15 plays a regulatory role in the -S response in Arabidopsis thaliana seedlings. Multiple molecular and physiological responses to -S are attenuated in snrk3.15 mutants, including both early adaptive responses and later emergency salvage processes including nutrient deficiency induced senescence. When grown in soil with sufficient sulfur supply, snrk3.15 mutants showed no clear phenotypes, including no difference in seed sulfur content. Lastly, the proteome dataset generated from Col-0 and snrk3.15.1 Arabidopsis seedlings under -S conditions for this project is the first of its kind and will be a valuable research resource.

Durian (Durio zibethinus L.) is an important economic fruit crop in Southeast Asia that produces volatile sulfur compounds (VSCs) during fruit ripening. Methionine γ-lyase (MGL) is the key enzyme involved in the production of VSCs, such as methanethiol and ethanethiol, coinciding with the upregulation of durian MGL (DzMGL) during fruit ripening. Although the VSCs present in durian pulps have been extensively studied, the regulation of genes involved in VSC production in fruit has not yet been explored. Yeast one-hybrid screening of the promoter of durian MGL (pDzMGL) and ca. 1200 Arabidopsis transcription factors (TFs) together with a transcriptomics analysis identified candidate HD-ZIP TF families in durian. Here, dual-luciferase assays, in silico promoter analysis, and electrophoresis mobility shift assays were performed to confirm the interaction between pDzMGL and DzHD-ZIP1.8. The results revealed that the ethylene-responsive DzHD-ZIP1.8 localized in the nucleus, where it functioned as an activator and bound to pDzMGL to control its expression during ripening. Therefore, a TF that regulates the expression of DzMGL in durian fruit during ripening was identified in this study. Moreover, this study elucidated the molecular regulation of an important gene involved in VSC production, which affects consumer preferences.

Because plants are immobile, they have developed intricate mechanisms to sense and absorb nutrients, adjusting their growth and development accordingly. Sulfur is an essential macroelement, but our understanding of its metabolism and homeostasis is limited. LSU (RESPONSE TO LOW SULFUR) proteins are plant-specific proteins with unknown molecular functions and were first identified during transcriptomic studies on sulfur deficiency in Arabidopsis. These proteins are crucial hubs that integrate environmental signals and are involved in the response to various stressors. Herein, we report the direct involvement of LSU proteins in primary sulfur metabolism. Our findings revealed that the quadruple lsu mutant, q-lsu-KO, which was grown under nonlimiting sulfate conditions, exhibited a molecular response resembling that of sulfur-deficient wild-type plants. This led us to explore the interactions of LSU proteins with sulfate reduction pathway enzymes. We found that all LSU proteins interact with ATPS1 and ATPS3 isoforms of ATP sulfurylase, all three isoforms of adenosine 5´ phosphosulfate reductase (APR), and sulfite reductase (SiR). Additionally, in vitro assays revealed that LSU1 enhances the enzymatic activity of SiR. These results highlight the supportive role of LSU proteins in the sulfate reduction pathway.

The transcription factor Sulfur Limitation 1 (SLIM1) belongs to the plant-specific Ethylene Insenstive3-Like transcription factor family and is known to coordinate gene expression in response to sulfur deficiency. However, the roles of SLIM1 in nutrient-sufficient conditions have not been characterized. Employing constitutive SLIM1 overexpression (35S::SLIM1) and CRISPR/Cas9 mutant plants (slim1-cr), we identified several distinct phenotypes in nutrient-sufficient conditions in Arabidopsis thaliana. Overexpression of SLIM1 results in plants with approximately twofold greater rosette area throughout vegetative development. 35S::SLIM1 plants also bolt earlier and exhibit earlier downregulation of photosynthesis-associated genes and earlier upregulation of senescence-associated genes than Col-0 and slim1-cr plants. This suggests that overexpression of SLIM1 accelerates development in A. thaliana. Genome-wide differential gene expression analysis relative to Col-0 at three time points with slim1-cr and two 35S::SLIM1 lines allowed us to identify 1,731 genes regulated directly or indirectly by SLIM1 in vivo.

Brassica species contains sulfur-containing secondary compounds including glucosinolates which might protect plants from pathogens. In the present investigation, the first leaves of Brassica rapa were grown in different situations such as sulfate-sufficient and deprived conditions, and infected with two types of fungi namely, Alternaria brassicicola and Botrytis cinerea as the specialist Brassica pathogen and generalist pathogen, respectively. The glucosinolates level was locally increased mainly due to indolic glucosinolates when the plant was infected with both fungi. This increase was in line with the increase in the expression of the genes including CYP79B2, CYP79B3, and CYP83B1 which are responsible for the biosynthesis of indolic glucosinolates and their regulation (MYB34 and MYB51). However, the locally induced indolic glucosinolates in plants infected with A. brassicicola were substantially higher than those of the plants infected with B. cinerea. The expression of the genes responsible for the biosynthesis of indolic glucosinolates was increased by infection of plant with A. brassicicola. The increase in the content of indolic glucosinolate occurred in the second leaf and roots, demonstrating a systemic response to fungal infection. Upon infection of plants with fungi, the content of both glucosinolates was reduced, while the expression of the most genes responsible for the biosynthesis of indolic glucosinolates was enhanced in plants infected with A. brassicicola. This may indicate that indolic glucosinolates are important in response to necrotrophic fungi in Brassica.

Durian (Durio zibethinus L.), an economically important fruit crop in Southeast Asia, is known for its strong sulfuryl aroma resulting from the accumulation of sulfur-containing compounds. However, our understanding of sulfur metabolism in fruit remains limited. In this study, we focused on the functional characterization of durian methionine γ-lyase (DzMGL), the key enzyme in volatile sulfur compound (VSC) production. In addition, to gain a better insight into sulfur metabolism, we profiled metabolites related to this process using various methods and assessed the expression of relevant genes through reverse transcription-quantitative PCR. Our gene and metabolite analyses revealed that sulfur metabolism is activated during ripening. Interestingly, DzMGL exhibited a higher efficiency in using L-cysteine than Arabidopsis MGL. The differential levels of the produced VSCs explained the aroma intensity-related differences between the two commercial Thai cultivars 'Chanee' and 'Monthong'. Furthermore, the cysteine availability-related differences between the two cultivars could contribute to this variation, with 'Chanee' containing higher cysteine levels than 'Monthong'. Our findings indicated that γ-glutamylcysteine was the preferred form of sulfur storage in durian pulps, with a potential recycling process providing intermediates that flux methionine toward ethylene biosynthesis and VSC production. This study provides novel insights into sulfur metabolism in durian fruit.

Wind is an environmental stimulus that stresses plants of all growth forms at all life-stages by influencing the development, architecture, and morphology of roots and shoots. However, comparative studies are scarce and no study directly investigated whether shoot and root morphological traits of trees, grasses and forbs differ in their response to short wind pulses of different wind intensity. In this study, we found that across species, wind stress by short wind pulses of increasing intensity consistently changed root morphology, but did not affect shoot morphological traits, except plant height in four species. Wind effects in roots were generally weak in tree species but consistent across growth forms. Furthermore, plant height of species was correlated with changes in specific root length and average diameter.Our results indicate that short-pulse wind treatments affect root morphology more than shoot morphology across growth forms. They further suggest that wind stress possibly promotes root anchorage in young plants and that these effects might depend on plant height.

Under conditions of sulfur deprivation, O-acetylserine (OAS) accumulates, which leads to the induction of a common set of six genes, called OAS cluster genes. These genes are induced not only under sulfur deprivation, but also under other conditions where OAS accumulates, such as shift to darkness and stress conditions leading to reactive oxygen species (ROS) or methyl-jasmonate accumulation. Using the OAS cluster genes as a query in ATTED-II, a co-expression network is derived stably spanning several hundred conditions. This allowed us not only to describe the downstream function of the OAS cluster genes but also to score for functions of the members of the co-regulated co-expression network and hence the effects of the OAS signal on the sulfate assimilation pathway and co-regulated pathways. Further, we summarized existing knowledge on the regulation of the OAS cluster and the co-expressed genes. We revealed that the known sulfate deprivation-related transcription factor EIL3/SLIM1 exhibits a prominent role, as most genes are subject to regulation by this transcription factor. The role of other transcription factors in response to OAS awaits further investigation.

Senescence is a highly regulated process driven by developmental age and environmental factors. Although leaf senescence is accelerated by nitrogen (N) deficiency, the underlying physiological and molecular mechanisms are largely unknown. Here, we reveal that BBX14, a previously uncharacterized BBX-type transcription factor in Arabidopsis, is crucial for N starvation-induced leaf senescence. We find that inhibiting BBX14 by artificial miRNA (amiRNA) accelerates senescence during N starvation and in darkness, while BBX14 overexpression (BBX14-OX) delays it, identifying BBX14 as a negative regulator of N starvation- and dark-induced senescence. During N starvation, nitrate and amino acids like glutamic acid, glutamine, aspartic acid, and asparagine were highly retained in BBX14-OX leaves compared to the wild type. Transcriptome analysis showed a large number of senescence-associated genes (SAGs) to be differentially expressed between BBX14-OX and wild-type plants, including ETHYLENE INSENSITIVE3 (EIN3) which regulates N signaling and leaf senescence. Chromatin immunoprecipitation (ChIP) showed that BBX14 directly regulates EIN3 transcription. Furthermore, we revealed the upstream transcriptional cascade of BBX14. By yeast one-hybrid screen and ChIP, we found that MYB44, a stress-responsive MYB transcription factor, directly binds to the promoter of BBX14 and activates its expression. In addition, Phytochrome Interacting Factor 4 (PIF4) binds to the promoter of BBX14 to repress BBX14 transcription. Thus, BBX14 functions as a negative regulator of N starvation-induced senescence through EIN3 and is directly regulated by PIF4 and MYB44.

Despite the fact that sulfolipids comprise a mere 4–7

Studies investigating the effect of aboveground herbivory on plants often use clipping to simulate the effects of herbivores, for practical reasons. However, herbivore movements and transfer of oral secretions during herbivory may cause a different response in plant physiology and morphology compared to clipping. While studies have compared effects of real herbivory vs. clipping on biomass production, plant physiology, and shoot morphology, no study has compared such effects on root morphology. Therefore, we investigated the effect of herbivory by grasshoppers, herbivory simulated by clipping, and no herbivory on root morphological traits of ten grassland plant species. Root morphological traits were differently affected by the two herbivory treatments. Grasshopper herbivory significantly changed root morphology toward thinner roots with increased specific root length and root area, and decreased root tissue density compared to untreated control plants. Clipping had mostly similar, but weaker effects on root morphology than grasshopper herbivory. On the species level, grasshopper herbivory led to strongest changes in root morphology in almost all cases. In contrast, depending on the species, clipping resulted in varying root morphological trait values similar to grasshopper-damaged plants, or in some cases, more closely aligned with control plants. Though clipping was partly able to mimic the effects of herbivory by grasshoppers, results also indicate that, depending on the species, grasshopper herbivory had different but mostly stronger effects. We, therefore, recommend that future studies apply herbivory with real herbivores to better reflect natural responses in plants and related processes that root morphological traits mediate.

The homeostasis of major macronutrient metabolism needs to be tightly regulated, especially when the availability of one or more nutrients fluctuates in the environment. Both sulfur metabolism and glucose signaling are important processes throughout plant growth and development, as well as during stress responses. Still, very little is known about how these processes affect each other, although they are positively connected. Here, we showed in Arabidopsis that the crucial transcription factor of sulfur metabolism, SLIM1, is involved in glucose signaling during shortage of sulfur. The germination rate of the slim1_KO mutant was severely affected by high glucose and osmotic stress. The expression of SLIM1-dependent genes in sulfur deficiency appeared to be additionally induced by a high concentration of either mannitol or glucose, but also by sucrose, which is not only the source of glucose but another signaling molecule. Additionally, SLIM1 affects PAP1 expression during sulfur deficiency by directly binding to its promoter. The lack of PAP1 induction in such conditions leads to much lower anthocyanin production. Taken together, our results indicate that SLIM1 is involved in the glucose response by modulating sulfur metabolism and directly controlling PAP1 expression in Arabidopsis during sulfur deficiency stress.

SUMMARYArabidopsis thaliana sulfur deficiency‐induced 1 and sulfur deficiency‐induced 2 (SDI1 and SDI2) are involved in partitioning sulfur among metabolite pools during sulfur deficiency, and their transcript levels strongly increase in this condition. However, little is currently known about the cis‐ and trans‐factors that regulate SDI expression. We aimed at identifying DNA sequence elements (cis‐elements) and transcription factors (TFs) involved in regulating expression of the SDI genes. We performed in silico analysis of their promoter sequences cataloging known cis‐elements and identifying conserved sequence motifs. We screened by yeast‐one‐hybrid an arrayed library of Arabidopsis TFs for binding to the SDI1 and SDI2 promoters. In total, 14 candidate TFs were identified. Direct association between particular cis‐elements in the proximal SDI promoter regions and specific TFs was established via electrophoretic mobility shift assays: sulfur limitation 1 (SLIM1) was shown to bind SURE cis‐element(s), the basic domain/leucine zipper (bZIP) core cis‐element was shown to be important for HY5‐homolog (HYH) binding, and G‐box binding factor 1 (GBF1) was shown to bind the E box. Functional analysis of GBF1 and HYH using mutant and over‐expressing lines indicated that these TFs promote a higher transcript level of SDI1 in vivo. Additionally, we performed a meta‐analysis of expression changes of the 14 TF candidates in a variety of conditions that alter SDI expression. The presented results expand our understanding of sulfur pool regulation by SDI genes.

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.

This article comments on: Henriet C, Balliau T, Aime D, Le Signor C, Kreplak J, Zivy M, Gallardo K, Vernoud V. 2021. Proteomics of developing pea seeds reveals a complex antioxidant network underlying the response to sulfur deficiency and water stress. Journal of Experimental Botany 72, 2611–2626.

Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under -S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency.

Durian is an economically important fruit of Southeast Asia. There is, however, a lack of in-depth information on the alteration of metabolic networks during its ripening. Here, we annotated 94 ripening-associated metabolites from the pulp of durian cv. Monthong fruit at unripe and ripe stages, using capillary electrophoresis- and gas chromatography- time-of-flight mass spectrometry, specifically focused on flavor-related metabolites. During ripening, sucrose content was found to be increased. Change in raffinose-family sugars is reported herein for the first time. The contents of malate and succinate increased, while those of citrate, an abundant organic acid, were unchanged. Notably, most amino acids increased, including isoleucine, leucine, and valine, whereas aspartate decreased, and glutamate was unchanged. Furthermore, transcriptome analysis was performed to support the dynamic changes in several flavor-related pathways, and for the identification of key candidate genes. Taken together, our results could be exploited for developing durian metabolic/genetic markers in the future.

Metabolites influence flowering time, and thus are among the major determinants of yield. Despite the reported role of trehalose 6-phosphate and nitrate signaling on the transition from the vegetative to the reproductive phase, little is known about other metabolites contributing and responding to developmental phase changes. To increase our understanding which metabolic traits change throughout development in Arabidopsis thaliana and to identify metabolic markers for the vegetative and reproductive phases, especially among individual amino acids (AA), we profiled metabolites of plants grown in optimal (ON) and limited nitrogen (N) (LN) conditions, the latter providing a mild but consistent limitation of N. We found that although LN plants adapt their growth to a decreased level of N, their metabolite profiles are strongly distinct from ON plant profiles, with N as the driving factor for the observed differences. We demonstrate that the vegetative and the reproductive phase are not only marked by growth parameters such as biomass and rosette area, but also by specific metabolite signatures including specific single AA. In summary, we identified N-dependent and -independent indicators manifesting developmental stages, indicating that the plant's metabolic status also reports on the developmental phases.