Renato Saleri

Keywords: digital generative tools, formal grammars, 3D modeling

Creative processes in today architecture and design are often accompanied today by a complete set of assistance tools inspired by biomimetic heuristics. Based on processes that took billions of years to set up, some formalisms now allow us to quickly explore vast areas of solutions. For example, they are able to optimize the optimum of a function in a reasonable time when there is no exact method or when the constraints given at an input are antagonistic. When not in a logic of multi-criteria optimization, one can also look for, among the innumerable creative supports offered by geometry or mathematics, those that will be best able to accompany the designer during the initial phases of conceptual exploration. We could of course rely on purely combinatorial logics while being sure to find -among the infinite possibilities offered by a universe number for example (Π, √2…) -a solution to our problem but in temporalities that would go far beyond the life of the cosmos itself. Among the experiments conducted by the MAP-Aria laboratory for nearly 20 years, many have relied on bio-inspired heuristics, others have tested combinatorial logic -without much hope -and finally some combined the most recent advances in Computer Vision and shape recognition to try to understand the artist's gesture and the notion of reproducibility or ownership of an artwork. These crucial questions consider the essence of the creative process itself and search the remaining place of the human being in the reduced free space left by the "thinking machines" of today and tomorrow.

Context.

Over the past twenty years, the considerable growth of digital tools has enabled the emergence of technologies capable of imitating and reproducing human behavior in an increasingly autonomous way. Initially conceived as artifacts capable of repeating tedious tasks over and over again in order to give humans complete freedom to focus on more interesting activities, our contemporary societies are seeing the emergence of a large number of tools capable of assisting us in our daily actions. They are now equipped with behavioral autonomy and are increasingly able to make decisions for us. We've come a long way since the first automatons, designed to reproduce a sequence of predetermined actions, and the devices capable today of potentially replacing humans cognitive faculties. But although the idea of "artificial intelligence" already emerged in the early 1950s in Alan Turing's now famous paper "Computing Machinery and Intelligence" (Turing, 1950), it is certain thateven today -one wonders whether a machine is really capable of thinking.

"Poetry is not only in the verb. The poetry of facts is even stronger. Objects that mean something and are arranged with tact and skill create a poetic fact." [START_REF] Corbusier | Le: Vers une architecture -Éditions Crès, Collection de "L'Esprit Nouveau[END_REF] 2 The fatal birth of architecture. The obligation of order [START_REF] Corbusier | Le: Vers une architecture -Éditions Crès, Collection de "L'Esprit Nouveau[END_REF].

Architects did not wait for computers to rely on formalisms able to accompany the creative process: "The regulator layout is an insurance against arbitrariness. It is mind soothing." [START_REF] Corbusier | Le: Vers une architecture -Éditions Crès, Collection de "L'Esprit Nouveau[END_REF] Beyond this assertion, which in our opinion frames the act of thinking a little too closely, it is true that not everyone can or wants to systematically resort to the "deregulation of the senses to reach the unknown" (from A. Rimbaud, letter to G. Izambard -1871: in French: "Il s'agit d'arriver à l'inconnu par le dérèglement de tous les sens"). "The regulator layout is a path; it is not a recipe. Its choice and its modalities of expression are an integral part of architectural creation [...] by establishing the preeminence of the initiatory trace, bearing the seed of the form and capable of guiding it in its slow maturation towards its finalization". [START_REF] Corbusier | Le: Vers une architecture -Éditions Crès, Collection de "L'Esprit Nouveau[END_REF] The deep harmony of a work cannot, however, be summed up in the beams that seem to fit happily around the guidelines of its composition, and besides "it would be quite possible, by multiplying the points of reference and without specifying the acceptable margin of error, to always find what one presupposes". [START_REF] Bouttier | L'étude des traces régulateurs de l'architecture médiévale in[END_REF].

We must understand that the regulator layout is a mediator between the abstract space of geometry and the concrete space of the project. Set aside the countless contemporary ramblings that force the function within the form, for the sake of the purity of the mathematical or geometrical expression source of its birth. Many of them flourish abundantly on the covers of fashionable magazines, but very few of them delight the users who borrow them. Moreover, one cannot be extremely sure either that the final project -however finite it may be -has reached its full maturity: one could only be happy with a state of "satisfactory incompletion" that best meets the programmatic and functional constraints defined at a given time, but that is another debate... Thus, if we consider N. Salingaros short essay "The visceral experience of architecture: object affordance and our need to grasp our surroundings", we can multiply the geometrical abstractions that will seduce the observer's gaze, but we will have to look elsewhere for the rules of composition that will allow us to satisfy our need for inner order: "Human beings respond to spaces, surfaces, detail, and ornament viscerally, which determines how a built structure will actually be used independently of whatever the architect intended." [START_REF] Salingaros | The visceral experience of architecture: object affordance and our need to grasp our surroundings[END_REF] But how to detach the reasoned perception of the architectural object from what could trigger a most profound accomplishment?

"By analogy, we can identify primary elements of architecture that are responsible for exerting the strongest influence of form and space on users. What we normally perceive as "architecture" is the surface of nested layers of a complex cognitive/response system. Less obvious but still primary (or primal) aspects of a building or a space influence our physiological and psychological responses: the success of a building or an urban space depends much more on these primal elements than on an intellectual analysis of its visible structure." [START_REF] Salingaros | The visceral experience of architecture: object affordance and our need to grasp our surroundings[END_REF] And, according to Rudofsky: "Without mentioning the first fifty centuries of its existence, historians present us with a well-ordered panorama of accepted and recognized architectures, which is as arbitrary a way of conceiving the art of building as if, for example, we were to trace the history of music back to the birth of the symphony orchestra. The forms of certain houses, sometimes handed down through a hundred generations, seem to be eternally valid, as are the forms of the basic tools." [START_REF] Rudofsky | Architecture sans architects[END_REF] Let us here quote Voltaire who, with his famous sentence: "Never use a new word unless it has these three qualities: necessary, intelligible, and sonorous", denounces the gratuitous substitution of a word of use by another word which would have only the merit of novelty: "it is not enriching the language," he says, "it is spoiling it."

In architectural design, creativity is both a myth and a taboo. For a long time, many researchers have been interested in the inadequacy of design assistance tools in terms of creativity and autonomy. To quote J. P. Chupin who himself invokes the work of D. Shön: "...architects are far from paying equal attention to process and product. If the introduction of information technology does not certainly increase the architect's creativity in his mission, most CAD software behaves like over-equipped drawing assistants: they presuppose both the maturity of the designer and that of the object of his design. To make full use of the potential of digital tools it is not enough to increase their ability to simulate materiality, but at the same time it is important to take over the relationship with the body they anaesthetize" [START_REF] Chupin | Escalade analogique et plongée numérique" Entre l'atelier tectonique et le studio virtuel dans l'enseignement du projet -pp 21 à 28[END_REF] The essence of the black box at the origin of the creative process cannot thus be altered other than by the mobilization of "situations to think", the only ones capable of stimulating the creative process by "successive jumps of intuition". Again, according to D. Shön : "This does not mean that computers are of no use, no assistance in design. Instead, we suggest that research should focus on computer environments that increase the designer's ability to capture, store, manipulate, organize and reflect on what they see." [START_REF] Chupin | Escalade analogique et plongée numérique" Entre l'atelier tectonique et le studio virtuel dans l'enseignement du projet -pp 21 à 28[END_REF] Beyond cognitive faculties, a question that arises today concerns precisely the ability of an artificial system to assist us in "creative" disciplines. Without wishing to supplant inspiration, we are now seeing the emergence of many tools capable of accompanying conceptual exploration, a fragile phase if ever there was one, because it comes from a set of cognitive processes that would be able to understand and produce an indefinite number of new processes. Serendipity, which is frequently used in the creative context to designate a form of intellectual availability, fortuitously brings rich teachings from unexpected discoveries or errors. Moravec's paradox establishes that often what is difficult in robotics is easier for man (and inversely, what is difficult for humans seems quite easy to computers…): we enter here into the dark space of a black box in which even the most optimistic predictions do not foresee an artificial intelligence supremacy before many decades. Let us consider instead the phenomena that are still poorly understood concerning the interpretation of intelligible data by the human brain, and in particular those with which, in our field, it is interesting to play. It is not a question of replacing creativity with an artificial cognitive process, but of understanding the levers by which a digital medium is capable of opening the doors of perception.

Malevitch's arkhitectons. From 1923 to the early 1930s, Kasimir Malevich produced several three-dimensional models, assemblages of abstract forms which appear similar to models of skyscrapers, called "arkhitektons". The drawings accompanying the construction of the models are called "planits". The arkhitektons are mostly white plaster models made up by several rectangular blocks added one to another. Usually a central bigger block is the main compositional element and smaller parallelepipeds are progressively added to it. No function is shown or translated into form, the final shape being the pure result of assembling abstract masses vertically or horizontally. With their spatialization of abstraction and their formal non-objectivity, the arkhitektons embody Malevich effort to translate the suprematist principles of composition to three-dimensional forms and architecture.

"In a series of prismatic, quasi architectural sculptures (which he called 'Arkhitektons') [he] sought to demonstrate the timeless laws of architecture underlying the ever changing demands of function. (…) Malevich's Arkhitektons resemble early De Stijl compositions in which ornament is non-figural and 'form' and 'ornament' are differentiated only by scale.

These studies are purely experimental and the buildings have no function and no internal organization. ". [START_REF] Colquhoun | Modern Architecture[END_REF] Responding to the issues stated by Malevitch in his supremacist manifesto, these formal games not only appeal to interpretative shifts due to their plastic ambiguity but also herald -perhaps unintentionally -the rise of those recursive formalisms that today are called "fractals".

"At the same time, we are discovering that traditional wisdom embedded in the built environment contains many of the design answers we now seek. Our ancestors who built towns and cities had an intuitive idea of which environments were more accommodating emotionally, and more healing (Alexander, 1979;Alexander et al., 1977). The tools they used to evaluate them were their own direct senses." [START_REF] Salingaros | The visceral experience of architecture: object affordance and our need to grasp our surroundings[END_REF] The tools we manipulate for conceptual support should therefore be more attuned to the deep resonances of our sensory biology. We can sense that juxtaposed relative quantities or judiciously superimposed tracings are at the origin of unexpected perceptive phenomena -such as optical illusions for example -or visually satisfying phenomena; this perception seems to be universally shared by all observers since time immemorial. Certain harmonic relationships, whether musical or geometric, arouse common feelings in most human beings and it has been shown that certain sensory stimuli seem to favour the development of plant species.

The observation of the growth phenomena of living organisms can thus largely inspire generative processes designed to be attuned to their ecosystem, whether natural or artificial. In this, we can draw on the cumulative experience of thousands of generations that have succeeded in adapting their evolution to the constraints of the environment in which they evolved. Is it not a universal mechanism to be able to adapt to the environment around us? Bjarke Ingels, from the BIG agency, sees the possibility of a kind of "architectural Darwinism" emerging within the decision-making groups that bring the idea to its concrete conclusion. Let us broaden the field of application of this fundamental notion and imagine that all the solutions generated by humanity to respond to the constraints linked to its evolution are evaluated, cross-referenced, hybridized or discarded before being adopted and put to the test of time.

"Long before the advent of architects, architecture emerged from the place, the time and what you have on hand to build it. It is more a form of community production, not the product of a few intellectuals or specialists, but of the spontaneous and continuous activity of a whole people, custodian of a common heritage and obedient to the lessons of a common experience." [3]

More interestingly, the survival of an idea will depend on its capacity to undergo during its existence a set of transformations likely to harmonize its functioning with the transformation of uses. If we want to generalize this concept, we could say that this is valid with regard to the "phenotype" -an object, a form or a building capable of surviving and "reproducing" (being imitated, or even copied) -as much as the "genotype" that is at the origin of it: an idea, a function or a path of thought or debate that remains and remains applicable whatever the circumstances. The very persistence of an aesthetic expression -a baroque and emergent expression of form -may even be at the origin of its survival when its functional support has disappeared. All this generates an infinity of persistent figures and signs that in turn shape our predisposition to find in them a subjectively personal and familiar meaning.

Bio-mimetic processes and optimization of generative strategies.

Combinatorial strategies, although capable of creating an infinity of formal solutions, are not able to produce interesting solutions in a temporality compatible with human existence. According to this point, current research focuses on those mechanisms that have regulated the generative processes of our biosphere for billions of years. Some of them were theoretically described several decades ago and still constitute today a solid exploratory basis for the ongoing investigations in this very field. For example, L-systems -described during the early 60' by the Hungarian biologist, Aristide Lindenmayer -make it possible to model in space and time some growth phenomena that mimic the growth dynamics involved, e.g., in plants evolution.

"Designers long have dreamed of buildings that behave like living things. Frank Lloyd Wright defined "organic architecture" as "building the way nature builds." In 1963, Archigram envisioned a "Living City"-community as organism. And now the Cascadia Green Building Council has issued a Living Building Challenge as the next stage of evolution toward "true sustainability." The challenge: "Imagine a building designed and constructed to operate as elegantly and efficiently as a flower." [START_REF] Hosey | Automatic Architecture: The ultimate goal of green building? How about automation? In architectmagazine www[END_REF] The recursive and auto-similar properties of their structure will allow them to be displayed with incremental levels of detail able to produce evolutionary shapes that model, for example, the transformation of an architectural ensemble built over time. L-systems are based on axioms and rule mechanisms whose formal expression is easily applicable to constructed elements with redundant and self-mimetic characteristics that actually imitates evolutionary process with a recursive regeneration of auto-similar patterns. Experience shows that the structured use of shape variables with a proper set of geometric transformations make it a very efficient 2D and 3D generator. Applied to topological germs specific to architecture, an infinite number of formal varieties can be obtained. Ongoing experiments aim to demonstrate the formal versatility of this model in generating the most disparate morpho stylistic varieties.

"But how does a flower grow ? It might be time to shift the conversation from product to process. What if buildings could be created in the same way a cell develops into a plant -from the bottom up instead of the top down? Technology may point the way. Automated processes are changing every aspect of design and construction, and it's only a matter of time before self-assembly completely takes over." [START_REF] Hosey | Automatic Architecture: The ultimate goal of green building? How about automation? In architectmagazine www[END_REF] This formal distopia resonates with the issues related to the use of combinatorial formalisms that have interested our research since the beginning. Following the chimera described by Borges in his famous account "The Library of Babel", it is tempting to imagine a device capable -in the field of architecture -of sweeping away, according to a combinatorial logic, all past, present and future architectural production of mankind. Of course, we are not at all in the same generative paradigm and it is highly likely that the vocabulary elements involved in such a generator would exceed the 24 characters used to populate the Borges library. A very modest attempt was made by the MAP on the occasion of the first Lyon Architecture Biennale in 2017, during which we left a machine running for only 5 days during the event and which was responsible for randomly producing a mapped projection on the two pillars surrounding the reception area. During the exhibition, nearly 300,000 digital arkhitektons were produced in pairs, most of which went unnoticed, either because of the inattention of the spectators or because the projection took place at night during the closing of the venue.

"The 'un-authored' object idea; objects formed out of chance and accident, that are then noticed and valued in equivalent ways to the art object is theoretically interesting because it questions the technical relationship between seeing and creating, and more broadly the dynamic of the viewer and the artist." [START_REF] Mcloughlin | The Un-authored Object in generative art 2007 -Politecnico di[END_REF] We like the idea of these machines working tirelessly to produce images that no one will be able to see. A little in this logic, we have reproduced an artistic installation on Line that already dates back about ten years: a matrix of pixels turned on or off according to a binary logic; when a pixel has finished its cycle (turned on and off) the adjacent pixel changes state.

For practical reasons all pixels are disposed in a square matrix, the logic remaining the same for the "line" pixel as for the adjacent pixel. According to this very simple rule, the occurrence of the state change for each pixel of the matrix will depend on its position in the row and will follow an exponential temporality. Nevertheless, left alone, this system will sweep away all existing binary possibilities and will eventually produce some happier solutions beyond a universe of meaningless configurations. Following the same logic, we have developed and experimented with a tool displaying a numerical sequence in the form of grey pixels distributed on a matrix whose l/h ratio can be modified. This tool makes it possible to easily highlight the periodicity of a real number: the sensitivity of the eye to moiré phenomena makes it possible in this precise case to appreciate the repetition of the decimals by simply observing the image produced. By modifying the l/h ratio it is even possible to vary the aspect of the display and thus to look for more interesting configurations. Is there however a hidden order in the series of decimals that make up the substance of irrational numbers? We could certainly find, following the example of the quest of the Aleph according to Borges, the sequence of numbers forming any past, present and future Instagram© picture but still it would be necessary to locate it in the infinite sequence of PI decimals: we would not have more probabilities to find its location than to find the novel "Les Misérables" by Victor Hugo in the famous library mentioned previously.

Without looking so far, it is possible to find in the already mentioned L-Systems harmonic phenomena that occur after a large number of iterations. Are they due to the particular recursive properties of these formalisms on which all types of rules can be applied? To do this, it is necessary to act with great finesse on the factors applied recursively in order to observe unexpected alignments when the major axes of composition approach particular angular ratios: it is fascinating to note to what extent the recursive functions applied to geometry are capable of modelling the formal expression of a large number of more primitive plant and animal species. Is the initial search for adaptation to an unfamiliar environment simpler when one adopts star-shaped or symmetrical forms? This is an interesting avenue for architecture. Ernst Haeckel's plates artfully illustrate the impressive beauty of the biological world, a beauty that expresses itself in our eyes but which has no other reason in the end than to offer species the tools to survive and procreate.

Conclusion

Recalling Nature with efficient generative paradigms seems to be relevant to discriminate the exponential spread-out of possible solutions of un-controlled growth approaches. However, the drawback of such processes is in its unpredictability or its poor response to domains where it is hard or impossible to define a computational fitness function. Interactive Genetic Algorithms (IGA) or Aesthetic Selection uses human evaluation for the fitness function, typically when the form of fitness function is not known, such as visual appearance or aesthetics evaluation.

It is so possible to use well-established mechanisms that have been experimented by nature for billions of years and that have produced -needless to say -workable results in many areas. Well implemented in today's 3D tools, some inspired organic formalisms are now used in many fields: although they certainly do not deploy the same functional complexity as their living counterparts, they are extremely gifted at optimizing multicriteria problems, supervising monitoring operations or assisting in operational decision-making. As said before, it is no longer necessary to go through the tree of possibilities in its entirety, we will be able to make drastic shortcuts in the production of optimal solutions considering a set of constraints placed at the beginning and according to a time span more compatible with the duration of our own existence.

Fig 1 .

 1 Fig 1. Author's early L-System generator (2008).

Fig 2 .

 2 Fig 2. The Arkhology generator -Biennale d'architecture de Lyon.(2017).

Fig 3 .

 3 Fig 3. One amongst the 2 256 display possibilities of a 16x16 b&w px matrix.

Fig 4 .

 4 Fig 4. Combining a GA approach with a L-System generator (2020).


Hye-In Nam

Zaigham Shahzad

Yanniv Dorone

Sophie Clowez

Kangmei Zhao

Nadia Bouain

Huikyong Cho

Seung Y Rhee
email: srhee@carnegiescience.edu

Hatem Rouached

Interdependent Iron and Phosphorus Availability Controls Photosynthesis Through Retrograde Signaling

Iron deficiency hampers photosynthesis and is associated with chlorosis. We recently showed that iron deficiency-induced chlorosis depends on phosphorus availability. How plants integrate these cues to control chlorophyll accumulation is unknown. Here, we show that iron limitation downregulates photosynthesis genes in a phosphorus-dependent manner. Using transcriptomics and genome-wide association analysis, we identify two genes, a chloroplastic ascorbate transporter (PHT4;4) and a nuclear transcription factor (bZIP58), which prevent the downregulation of photosynthesis genes leading to the stay-green phenotype under iron-phosphorus deficiency. Joint limitation of these nutrients induces ascorbate accumulation by activating expression of an ascorbate biosynthesis gene, VTC4, which requires bZIP58. Exogenous ascorbate prevents iron deficiency-induced chlorosis in vtc4 mutants, but not in bzip58 or pht4;4. Our study demonstrates chloroplastic ascorbate transport is essential for preventing the downregulation of photosynthesis genes under iron-phosphorus combined deficiency. These findings uncover a molecular pathway coordinating chloroplast-nucleus communication to adapt photosynthesis to nutrient availability.

significantly reduced the accumulation of chlorophyll in all three species (Figure 1D). However, under -Fe-P conditions, chlorophyll content was comparable to control (+Fe+P) conditions in these species (Figure 1D). Next, we focused on Arabidopsis to gain insights into the physiological and molecular processes underlying the recovery of chlorosis under -Fe-P conditions. First, we asked whether absence of chlorosis under -Fe-P is caused by an increase of Fe levels in shoots. Plants grown in -Fe+P conditions decreased total Fe in shoots by 2-fold compared to +Fe+P conditions (Figure 1E). On the other hand, under +Fe-P conditions, Fe levels increased by 2.2fold relative to +Fe+P conditions (Figure 1E). Surprisingly, Fe levels in plants grown under -Fe-P were reduced and indistinguishable from the Fe levels in -Fe+P conditions (Figure 1E). Therefore, the lack of chlorosis under -Fe-P is unlikely to be caused by more Fe available in shoots. To further test this hypothesis, we assessed free Fe in leaves. Because FERRITIN1 (AtFER1) chelates Fe and its mRNA increases when Fe is in access, AtFER1 gene expression can be used as a read-out for intracellular Fe nutritional status [START_REF] Bournier | Arabidopsis ferritin 1 (AtFer1) gene regulation by the phosphate starvation response 1 (AtPHR1) transcription factor reveals a direct molecular link between iron and phosphate homeostasis[END_REF] . We thus quantified the expression of AtFER1 in shoots under replete or deficient Fe and P in the growth media. Consistent with the total Fe levels, AtFER1 expression was increased significantly under +Fe-P conditions relative to +Fe+P (Figure 1F). However, AtFER1 expression decreased under both -Fe+P and -Fe-P conditions relative to +Fe+P (Figure 1F). Interestingly, -Fe-P condition caused a slightly bigger reduction in AtFER1 expression than -Fe+P did, suggesting that there may be even less free Fe in -Fe-P conditions than in -Fe+P conditions. Taken together, these data show that the onset of chlorosis during -Fe requires sufficient P in the growth media, and that the "stay green" phenotype under the combined -Fe-P deficiency cannot be linked to Fe nutritional status in leaves.

To understand the cause of chlorophyll reduction in response to -Fe, we first explored the timing of -Fe sensing and photosynthetic function response. Since -Fe affects chlorophyll accumulation and photosystem II (PSII) activity [START_REF] Bertamini | Effect of iron deficiency induced changes on photosynthetic pigments, ribulose-1, 5-bisphosphate carboxylase, and photosystem activities in field grown grapevine (Vitis vinifera L. cv. Pinot noir) leaves[END_REF][START_REF] Liu | The ethylene response factor AtERF4 negatively regulates the iron deficiency response in Arabidopsis thaliana[END_REF] , we monitored their kinetics over 172 hours (h) (Figure 2A-B, Figure S1A-C). Arabidopsis plants were first grown on +Fe+P media for one week, and then transferred to +Fe+P, -Fe+P, or -Fe-P conditions. -Fe+P caused a significant decrease in chlorophyll content observable starting at 52 h after the transfer to -Fe+P (Figure 2A). However, transfer to -Fe-P did not affect chlorophyll content, even at 172 h after the transfer (Figure 2A). To determine how photosynthesis was affected, we measured Fv/Fm, preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

which reflects the quantum yield of photochemistry and is a measure of PSII activity [START_REF] Bertamini | Effect of iron deficiency induced changes on photosynthetic pigments, ribulose-1, 5-bisphosphate carboxylase, and photosystem activities in field grown grapevine (Vitis vinifera L. cv. Pinot noir) leaves[END_REF][START_REF] Liu | The ethylene response factor AtERF4 negatively regulates the iron deficiency response in Arabidopsis thaliana[END_REF] . Plants under -Fe+P decreased Fv/Fm observable starting at 52h, indicative of compromised electron transport through PSII, and which coincides with the decrease of chlorophyll accumulation (Figure 2B). By 172 h, PSII activity was substantially reduced under -Fe+P compared to +Fe+P. However, plants under -Fe-P showed slightly lower but stabilized Fv/Fm compared to those in +Fe+P (Figure 2B). These physiological characterizations showed that chlorophyll accumulation and PSII activity were affected by -Fe, and both responses were P-dependent (Figure 2A-B).

Based on these findings, we selected three time points at 39, 52, and 76 h after the transfer of plants to +Fe+P, -Fe+P, or -Fe-P to conduct a global gene expression analysis in shoots (Figure 2C, Figure S2A-F). We identified genes whose expression levels were either increased or decreased by -Fe+P relative to +Fe+P by at least 2fold at a p-value < 0.05 (Figures 2D, Table S1). Even more genes were either upregulated or downregulated in -Fe-P conditions relative to +Fe+P (Figures 2D, Figure S3A-C). A total of 671 and 2434 transcripts were uniquely differentially regulated in response to -Fe+P or -Fe-P, respectively, supporting the existence of different signaling pathways under the two conditions (Figure S 3A-C). To identify functions enriched in genes that were differently regulated by -Fe+P or -Fe-P, we performed Gene Ontology (GO) enrichment analysis. Genes specifically downregulated by -Fe-P but not by -Fe+P at 52 h and 76 h (52 genes; Figure S4A, Table S1) showed an enrichment for ribosomal genes (Figure S4B) while upregulated genes (162 genes, Figure S4C, Table S1) revealed an enrichment for genes involved in cation transport, response to water, and ester hydrolysis (Figure S4D, Table S1). On the other hand, GO analysis of the 32 genes specifically downregulated by -Fe+P but not affected by -Fe-P at 52 h and 76 h (Figure 2E, Table S1) revealed an enrichment of genes related to the chloroplast and photosynthesisrelated processes (Figure 2F), while upregulated genes (Figure S4E) were enriched for genes related to cellular respiration, oxidation-reduction process, and energy metabolism (35 genes; Figure S4F). Altogether, the transcriptomics analysis indicated that the control of chloroplast function is an integral component of the nuclear transcriptomic response to -Fe, which is dependent on P availability. We also learned that the photosynthesis-related phenotypes we observed under -Fe+P, but not under -Fe-P, could be due to downregulation of key photosynthesis regulators.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

To decode the signaling pathways that control expression of the photosynthesis genes in response to -Fe+P, we exploited natural variation in expression of the 32 genes that were down-regulated by Fe deficiency in a P-dependent manner in a worldwide collection of A. thaliana accessions [START_REF] Kawakatsu | Epigenomic diversity in a global collection of Arabidopsis thaliana accessions[END_REF] . One way to identify regulatory mechanisms could be to perform expression genome-wide association studies (eGWAS) using the expression level of individual genes across Arabidopsis accessions. Strikingly, we found that expression levels of the 32 genes are predominantly positively associated with each other across 727 Arabidopsis accessions (Figure S5A) [START_REF] Kawakatsu | Epigenomic diversity in a global collection of Arabidopsis thaliana accessions[END_REF] . We then performed Principal Component Analysis (PCA) to reduce the dimensionality of these expression data. PC1 explained 89.5% of the variation in expression of these genes across Arabidopsis accessions (Figure S5B).

The contribution of each accession to PC1 was then used to perform a genome-wide association study (GWAS) (Figure 3A). Our GWAS analysis detected 38 QTLs containing 145 candidate genes, based on a 20-kb window per QTL and using a 5% false discovery rate (FDR) threshold, with the highest peak (Chromosome 2) occurring in an intergenic region (Figure 3A). In this study, we followed up one of the QTLs that contained the inorganic phosphate transporter PHT4;4 (AT4G00370) (Figure 3A, Figures S6 andS7) given its role in ascorbic acid (AsA) transport into chloroplasts, which was proposed to be important for maintaining the xanthophyll cycle for dissipation of excessive light energy to heat in photosynthesis [START_REF] Miyaji | AtPHT4; 4 is a chloroplast-localized ascorbate transporter in Arabidopsis[END_REF] . To determine if PHT4;4 has any role in -Fe+P dependent chlorosis, we examined mutants with a null pht4;4 allele. Under -Fe+P, chlorophyll was significantly reduced in pht4;4 mutants, similar to wild type plants (Figure 3B-C). However, -Fe-P conditions failed to recover chlorosis and chlorophyll reduction in pht4;4 mutants, unlike wild type plants (Figure 3B-C). Introduction of the wild type PHT4;4 allele into a pht4;4 mutant background complemented these phenotypes (Figure 3B-C). The chlorotic phenotype of the pht4;4 mutant under -Fe-P suggested that transport of AsA into chloroplasts could be important for the "stay green" phenotype.

To test our hypothesis about the role of AsA in preventing chlorosis under -Fe-P conditions, we first assessed how Fe and P availabilities regulate the expression of VITAMIN C (VTC) enzymes involved in AsA biosynthesis in plants [START_REF] Torabinejad | VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants[END_REF] . Our RNA-seq analysis identified that -Fe-P caused a 2 to 3-fold increase in VTC1, VTC2, and VTC4 expression, which we confirmed using qRT-PCR (Figure 3D). However, -Fe in preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

the presence of P (-Fe+P) caused a 2-fold decrease in the mRNA abundance of VTC4 (Figure 3D). VTC4 is the final enzyme in the AsA biosynthesis pathway [START_REF] Torabinejad | VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants[END_REF] . This prompted us to test the effect of the absence of VTC4 on chlorophyll accumulation under -Fe+P and -Fe-P conditions. Under -Fe-P, mutants with a vtc4 null allele were still chlorotic, similarly to pht4;4 and in contrast to wild type plants (Figure 3B-C).

These data show that AsA contributes to preventing chlorosis in -Fe-P conditions.

Next, we tested whether the chlorotic phenotype is due to variations in AsA levels. In wild type, AsA levels decreased significantly under -Fe+P at 52 h after the transfer relative to control (+Fe+P), whereas no change was detected under -Fe-P (Figure 3E), suggesting that AsA levels were associated with -Fe-mediated chlorosis. To test whether AsA levels were associated with chlorosis in general, we measured AsA levels in AsA synthesis (vtc4) mutant plants. Under +Fe+P, vtc4 plants accumulated 35% less AsA than wild type plants, and AsA levels remained unchanged in response to -Fe+P or -Fe-P stress (Figure 3E). However, vtc4 plants did not show the chlorotic phenotype under +Fe+P, which indicated that the level of AsA contributed to the chlorotic phenotype specifically under -Fe and this contribution was dependent on P availability. In addition, the AsA transporter (pht4;4) mutants showed similar AsA levels as the wild type even though pht4;4 plants were still chlorotic in -Fe-P (Figure 3E). To determine whether AsA accumulation in the cell or its transport to the chloroplast is associated with the development of chlorotic phenotype in -Fe+P, we tested the effect of an exogenous supply of AsA in wild type, vtc4, and pht4;4 plants (Figure 3B-C). Exogenous AsA alleviated the chlorosis caused by -Fe+P in wild type and vtc4 mutant plants. However, pht4;4 mutants failed to stay green under -Fe+P+AsA conditions (Figure 3B-C), indicating that the transport of AsA to the chloroplast is required for -P mediated 'stay green' phenotype under Fe deficiency. Our results showed that -P prevents the downregulation of VTC4 by -Fe and associated changes in AsA accumulation, and that the PHT4;4-mediated transport of AsA to chloroplasts is required for the maintenance of chlorophyll content under combined deficiency of Fe and P.

We next asked whether PHT4;4-mediated AsA transport to the chloroplast is important for regulation of the photosynthesis-related genes that were specifically down-regulated by -Fe in a P-dependent manner. First, we tested the effects of preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

PHT4;4 inactivation on the expression of these photosynthesis related genes using qRT-PCR (Figure 4A). While -Fe+P significantly downregulated the mRNA abundance of these genes in wild type plants (Col-0), -Fe-P prevented this response (Figure 4A). Furthermore, adding AsA to -Fe+P mimicked -Fe-P response in preventing down-regulation of the photosynthesis genes. Under -Fe+P, pht4;4 mutant plants showed a decrease in the mRNA abundance of these genes comparable to wild type plants (Figure 4A). However, in contrast to the wild type, these genes were still downregulated in pht4;4 plants under -Fe-P as well as -Fe+P supplemented with AsA (Figure 4A). Taken together, these data indicate that the transport of AsA to chloroplasts via PHT4;4 is central to preventing the downregulation of photosynthesis-related genes under -Fe-P.

How does -Fe+P affect the expression of photosynthesis-related genes? To look for potential transcriptional regulators of these genes, we examined the candidate genes from the GWAS analysis. We found that bZIP58 (AT1G13600) (Figure 3A, Figure S6-S7), a putative transcription factor, underlies one of the strongest QTL peaks (Figure 3A). To test whether bZIP58 responds to Fe and P availability, we performed qRT-PCR of bZIP58 under various Fe and P availability. bZIP58 was strongly downregulated by -Fe, and this down-regulation depended on P availability (Figure 4B). In addition, bZIP58 was partially required to induce VTC4 expression under -Fe-P conditions (Figure 4C). This led us to examine the contribution of bZIP58 in regulating the -Fe+P specific photosynthesis-related genes under +Fe+P, -Fe+P, -Fe-P conditions (Figure 4A). Mutants with the bzip58 null allele showed a remarkable constitutive decrease in the expression of these 32 photosynthesis-related genes (Figure 4A). bZIP58 localizes to the nucleus (Figure 4D), which is consistent with a role as a transcription factor. Taken together, these findings support the idea that bZIP58 is a key regulator of photosynthesis-related genes, and its absence could alter chlorophyll accumulation regardless of Fe and P availability. Genetic inactivation of bZIP58 indeed causes a constitutive decrease in chlorophyll content, and the mutant line is chlorotic (Figure 4E-F). The expression of bZIP58 gene in bzip58 plants complements the constitutive chlorosis phenotype, and the complemented line responds to Fe and P availability similarly to wild type plants (Figure 4E-F). Furthermore, AsA supplementation could not rescue the chlorotic phenotype of bzip58 mutants (Figure 4E-F), indicating that pZIP58 lies downstream of AsA action.

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These data show that bZIP58 controls the expression of photosynthesis-related genes and is transcriptionally regulated in response to -Fe depending on P availability, likely by mediating the perception of AsA.

Based on our findings, we propose a model to explain how P availability modulates Fe deficiency-induced chlorosis (Figure 4G). -Fe+P causes a decrease in the expression of AsA biosynthesis gene VTC4 (Figure 3D). Exogenous AsA supply prevents the development of chlorosis in vtc4 under -Fe-P, but not in the pht4;4 that transports AsA to the chloroplast (Figure 3B). We thus propose that AsA deficiency in the chloroplast under Fe limited conditions triggers chlorosis. How does AsA in the chloroplast affect photosynthesis? AsA has an antioxidizing action that detoxifies reactive oxygen species (ROS) through its scavenging properties [START_REF] Noctor | Ascorbate and glutathione: keeping active oxygen under control[END_REF] , thus making ROS a potential signaling molecule [START_REF] Finkel | Signal transduction by reactive oxygen species[END_REF][START_REF] Shapiguzov | ROS-talk-how the apoplast, the chloroplast, and the nucleus get the message through[END_REF][21] capable of modulating the expression of photosynthesis-related genes through bZIP58. To test this hypothesis, we measured the relative amount of ROS accumulation in shoots of wild type, pht4;4 and bzip58 plants under various Fe and P availability. -Fe+P caused a 2-fold increase in ROS accumulation in shoots of wild type plants, which partially depended on P availability (Figure 4H). pht4;4 plants displayed comparable ROS accumulation to that of the wild type under +Fe+P and -Fe+P. However, pht4;4 plants accumulated significantly higher ROS than wild type plants under -Fe-P (Figure 4H). In addition, bzip58 mutant plants displayed a constitutive increase in ROS accumulation (Figure 4H). To check whether ROS in turn can regulate the expression of bZIP58, we quantified bZIP58 expression in response to foliar application of H 2 O 2 . ROS treatment caused a 4-fold decrease in bZIP58 transcript accumulation (Figure 4B). Collectively, our results support the idea that under simultaneous Fe and P deficiency, AsA accumulation in the chloroplast prevents chlorosis by modulating ROS levels that may control the expression of photosynthesis genes via a putative transcription factor bZIP58 (Figure 4G). How ROS acts as a retrograde signal to alter nuclear gene expression to control photosynthesis under Fe and P limitation remains to be determined, though we now have several molecular targets with which to explore this field. Modulation of the discovered pathway could have a direct impact on plant growth in the field by improving plant photosynthetic activity while reducing nutrient supply. each condition. The 32 genes that were decreased specifically in -Fe+P but not in -Fe-P relative to +Fe+P at 52h and 76h (highlighted in purple) were used to perform genome-wide association studies. E) A heatmap showing gene expression patterns of the 32 genes in -Fe+P and -Fe-P relative to control (+Fe+P) at 39h, 52h, 76h after the transfer. LogFC, log2 fold change. F) Gene Ontology analysis for the biological processes (GO-BP) for the 32 genes whose mRNA abundance was specifically decreased by -Fe+P. FDR = false discovery rate. Source data are provided as a Source Data file.

Supplemental tables

Table S1. Differentially expressed genes in response to iron and/or phosphorus deficiency in A. thaliana. Gene transcript levels were determined in shoots of Col-0 plants grown in control (+Fe+P) condition for 7 days and then transferred to -Fe+P, -Fe-P, or +Fe+P for 39h, 52h, or 76h.

Table S2. List of primers used in this study.

Materials and Methods

Plants and growth conditions

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Seeds of Arabidopsis thaliana wild type (ecotype Columbia, Col-0, CS60000) and knock-out mutant lines SALK_139877 (AT1G13570), SALK_150849 (AT1G13580), SALK_063177 (AT1G13590), N571881 (AT1G13600), SALK_087271 (AT1G13605), SALK_130208 (AT1G13607), SALK_023173 (AT1G13608), SAIL_1243_E04 (AT1G13609), SAIL_897_D11 (AT1G13610), N469134 (At4g00355), SALK_128714 (AT4G00360), N469134 (AT4G00370), SAIL_842_E09 (AT4G00380), N866595 (At4g00390), SAIL_633_E10 (At4g00400) and SALK_077222 (AT3G02870) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). Homozygous mutant lines were confirmed by PCR using the primers listed in Table S2 Rice (Oryza sativa cv Nipponbare) plants were grown hydroponically in 0.25X Yoshida media 24 under light/dark cycle of 14/10 h, and temperature of 28/25 °C.

Single (-P or -Fe) and combined (-P-Fe) nutrient deficiency stresses were applied to 10 day-old plants. NaH 2 PO 4 (0.33 mM) and Fe-NaEDTA (0.04 mM) present in the complete media were omitted in the P-and/or Fe-deficient media.

Iron concentration measurement

Arabidopsis seeds were germinated and grown in the control (+Fe+P) media for 7

days, and then transferred to +Fe+P, iron deficient (-Fe+P), phosphate deficient (+Fe-P), or iron and phosphate deficient (-Fe-P) conditions and grown for 7 additional days. Plants were harvested and shoot samples were dried at 70 °C for 3 days. Total iron was extracted by acid digestion in 1N nitric acid using MARSX (CEM) microwave digester. A 1:10 dilution of the digested material was used to quantify total iron with inductively coupled plasma-atomic emission spectrometry (ICP-OES).

Analysis of photosystem II activity

Photosystem II (PSII) activity was defined as the maximum quantum yield of the primary quinone acceptor PSII, which was estimated by the ratio of variable fluorescence (Fv) and maximal fluorescence (Fm) of the chlorophyll, Fv/Fm 25 .

Arabidopsis wild type (Col-0) seeds were germinated and grown in control (+Fe+P)

for 7 days then transferred to three different media: +Fe+P, iron deficient (-Fe+P), and iron and phosphate deficient (-Fe-P) conditions for 0 h (time of the transfer), 15 h, 28 h, 39 h, 52 h, 76 h, 100 h, 124 h, 148 h, and 172 h. Plates containing the seedlings were dark adapted for 30 minutes followed by a very short (160 µs) exposure to a blue measuring beam to determine the minimal fluorescence (F0). The intensity of the detecting and the continuous illumination used was of 156 µE m -2 s -1 .

A saturating light flash (2600 µE m -2 s -1 , 250 ms) was applied to measure the maximum fluorescence (Fm). Kinetics were normalized to the maximum fluorescence (Fm). The maximum quantum yield of Photosystem II (Fv/Fm = (Fm -F0)/Fm) was measured for each growth condition 25 .

Chlorophyll content measurement

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Seeds of Arabidopsis genotypes were germinated and grown in control (+Fe+P) media for 7 days then transferred to three different media: +Fe+P, iron deficient (-Fe+P), and iron and phosphate deficient (-Fe-P) conditions. Fresh leaves (~30mg)

were incubated in 2.5mL of 80% acetone overnight in the dark at 4°C. Total chlorophyll content was measured using a UV-VIS spectrophotometer (Beckman Coulter, DU 530). The absorbance of the supernatant was measured at 645 nm and 633 nm. The concentration of total chlorophyll, chlorophyll a, and chlorophyll b were calculated as described previously 26 .

Ascorbic acid content determination

Seeds of Arabidopsis genotypes were germinated and grown in control (+Fe+P) media and then transferred to +Fe+P, -Fe+P, or -Fe-P media for 76h. Ascorbic acid (AsA) content was measured by a colorimetric assay as described previously 27 .

Briefly, shoots were collected and homogenized in ice-cold 6% trichloroacetic acid (TCA) (Sigma Aldrich). In the supernatant, Fe3+ (ferric ion) is reduced by AsA to the Fe2+ (ferrous ion) that, when coupled with 2,2-dipyridyl, forms a complex with a characteristic absorbance at 525 nm. A standard curve was generated using known concentrations of AsA made in 6% TCA to determine the AsA concentration. Blanks were prepared using only 6% TCA. AsA concentration was expressed as µmol g -1 fresh weight.

Hydrogen peroxide quantification

Seeds of Arabidopsis genotypes were germinated and grown in control (+Fe+P) media and then transferred to +Fe+P, -Fe+P, or -Fe-P media for 76h. Hydrogen peroxide (H 2 O 2 ) (Sigma-Aldrich) was quantified as described previously 28,29 . Fresh shoot tissues (0.2 g) were homogenized with 0.1% (w/v) TCA and were centrifuged at 12,000 g for 15 min at 4 °C. 0.5 ml of supernatant was added to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide. The absorbance of the reaction mixture was measured at 390 nm. The amount of H 2 O 2 was calculated using a standard curve prepared from known concentrations of H 2 O 2 ranging from 0.1 to 1 mM.

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+Fe+P, Fe+P, or -Fe-P. Shoot tissues were collected at 76h after the transfer, and then used for total RNA extraction as described in 33 . Each experiment was conducted with 16 plants and 4-6 plants were pooled for RNA extraction, resulting in 3-4 biological replicates. Two μg of the total RNA was used for reverse transcription (Promega) to synthesize cDNA using oligo(dT) primer (Promega). Real-time quantitative reverse-transcription PCR (qRT-PCR) was performed as described in 33 using 384-well plates with a LightCycler 480 Real-Time PCR System (Roche diagnostics). The Ubiquitin 10 mRNA (UBQ10: At4g05320) was used as control to calculate the relative mRNA level of each gene. The primers used in this study are listed in Table S2.

Genome wide association studies (GWAS)

Gene expression data of the 32 genes that were specifically downregulated by -Fe+P but not by -Fe-P relative to +Fe+P were downloaded from leaf expression data of 727 Arabidopsis accessions [START_REF] Kawakatsu | Epigenomic diversity in a global collection of Arabidopsis thaliana accessions[END_REF] . Normalized RNA-seq read counts of these genes were used to perform Principal Component Analysis, and contributions of the accessions to PC1 that explained 89.5% of the expression variance of the 32 genes were used to run genome-wide association (GWA) analysis. GWA mapping was performed using 1001 genomes SNP data 34 as implemented in the web application GWAPP 35 .

Bonferroni correction (α = 0.05) and false-discovery rate (FDR) at 5% 36 were implemented to account for multiple hypothesis tests.

Statistical analysis

Box plots were generated using a web based application "BoxPlotR" 37 . Statistical analyses of the data were performed using analysis of variance (ANOVA). One-way ANOVA with a Duncan post-hoc test, and two-way analysis of variance (ANOVA) and Tukey's honest significant difference (HSD) test were used to compare mean values.

For all the statistical analyses, the difference was considered statistically significant when the test yielded a p-value < 0.05.

Gene func>on Number of genes per gene func>on

E-21 E-21 E-20 E-11 E-11 E-7 E-7 E-2 E-8 E8 E10 AT5G04950 AT4G38100 AT5G20160 AT3G04770 AT1G70600 AT1G67260 AT1G74870 AT3G10113 AT5G50950 AT4G25630 AT3G19850 AT3G57490 AT3G23940 AT5G59680 AT2G21220 AT2G27840 AT1G77870 AT2G27710 AT3G44900 AT3G47370 AT4G38850 AT5G27120 AT5G61610 AT1G18330 AT4G33220 AT2G15810 AT5G67510 AT4G22020 AT5G13490 AT5G62440 AT2G32220 AT4G04940 AT3G18130 AT1G78206 AT1G48630 AT5G59850 AT5G11160 AT1G61580 AT5G01330 AT1G31835 AT5G44420 AT3G50825 AT1G53542 AT1G78090 AT2G32270 AT3G44750 AT5G62920 AT2G05914 AT3G48290 AT4G26780 AT5G61160 AT1G55205
Gene func>on FDR

2 E-3 2 E-3 2 E-3 2 E-3 1 E-2 1 E-1 1 E-1 2 E-1 2 E-1 2 E-1
C AT1G09530 AT3G10185 AT3G13080 AT3G05030 AT4G17470 AT1G55110 AT5G17165 AT3G08510 AT2G14878 AT4G37220 AT3G11580 AT5G06300 AT1G09350 AT5G62160 AT5G24770 AT1G30250 AT3G29784 AT4G27260 AT1G01250 AT3G46590 AT3G07350 AT5G66880 AT1G66390 AT3G59884 AT1G56650 AT5G40910 AT1G73325 AT5G50800 AT3G47220 AT2G36390 AT1G24130 AT1G22070 AT2G40750 AT4G19880 AT4G34310 AT1G71520 AT3G04210 AT1G56710 AT4G36350 AT3G49580 AT5G48850 AT1G25550 AT1G19630 AT4G28040 AT2G37980 AT3G52180 AT2G42530 AT5G62162 AT3G13277 AT1G51090 AT2G33380 AT1G26762 AT2G39800 AT1G23200 AT5G57190 AT5G51050 AT3G26220 AT1G50590 AT1G67610 AT1G67645 AT5G24155 AT1G08170 AT2G43530 AT1G35720 AT1G48370 AT4G19690 AT2G18050 AT5G53420 AT1G66110 AT5G10740 AT1G52000 AT5G59670 AT1G53310 AT3G04360 AT3G26510 AT3G11230 AT5G13820 AT3G02980 AT4G28260 AT5G43300 AT1G25370 AT2G16367 AT1G04330 AT5G64700 AT2G46600 AT1G76430 AT1G08920 AT4G24450 AT1G53170 AT5G17860 AT3G26740 AT5G08030 AT1G52800 AT3G08860 AT1G51140 AT3G57157 AT4G16540 AT2G32150 AT3G18080 AT5G40310 AT1G80120 AT3G50350 AT2G46270 AT5G66400 AT5G09440 AT5G55970 AT1G63530 AT2G42540 AT3G04350 AT3G25760 AT3G03530 AT5G14565 AT1G21529 AT3G54700 AT4G17900 AT1G18870 AT2G17280 AT4G19390 AT5G67080 AT4G35190 AT3G14700 AT1G05100 AT2G24000 AT4G16870 AT4G28140 AT5G24600 AT5G64000 AT1G21550 AT5G17340 AT2G47050 AT2G18670 AT4G08570 AT2G30540 AT5G24780 AT4G14020 AT5G15500 AT1G74810 AT1G20735 AT2G26620 AT4G16000 AT1G22220 AT5G09800 AT5G43150 AT4G19960 AT3G59300 AT4G21680 AT3G46970 AT1G61800 AT3G47820 AT4G23700 AT1G67860 AT1G52940 AT3G52310 AT4G03380 AT5G14330 AT3G59880 AT5G01880 AT1G69870 AT5G54980 AT4G36648 AT1G07985
-Fe+P -Fe-P 39 52 76 39 52 76 AGI D AT1G32520 -0.13 0.32 0.13 -0.03 -0.12 0.09 -0.10 0.39 0.15 -0.01 0.34 0.04 0.29 0.38 -0.07 -0.07 0.29 0.00 0.33 0.32 0.27 0.34 0.60 0.26 0.38 0.00 -0.11 0.20 0.29 0.63 0.21 AT1G50110 -0.18 -0.05 0.16 0.01 -0.01 0.04 0.09 -0.01 0.36 0.22 -0.03 0.30 -0.27 0.03 -0.05 -0.18 -0.07 -0.03 -0.12 0.14 0.16 -0.05 0.11 0.04 -0.15 0.10 -0.04 -0.08 0.05 0.14 AT2G39270 0.26 -0.08 -0.08 0.08 -0.07 0.12 0.00 -0.08 0.06 0.00 -0.02 0.27 0.03 -0.09 0.50 0.37 0.42 0.31 -0.05 0.09 0.37 0.11 0.13 0.07 -0.12 -0.01 0.47 0.11 0.11 AT1G64360 -0.06 -0.10 -0.04 0.01 0.09 0.04 -0.10 0.14 -0.05 0.14 0.14 0.03 0.01 0.21 0.05 0.16 0.19 -0.01 0.06 0.15 0.21 0.09 0.05 -0.04 -0.10 0.33 -0.01 0.18 AT3G16120 0.01 -0.03 0.03 0.19 0.01 0.05 -0.02 0.02 0.01 -0.02 -0.08 -0.01 -0.10 -0.10 -0.04 -0.04 0.12 0.06 -0.05 -0.06 -0.01 -0.07 0.11 -0.09 -0.06 0.13 -0.02 AT4G36060 0.15 0.17 -0.14 0.00 0.27 -0.06 0.03 -0.05 0.14 0.18 0.31 -0.10 0.06 0.09 0.03 -0.07 -0.03 -0.15 -0.02 0.01 0.07 0.21 0.07 -0.16 -0.10 -0.09 AT2G44940 0.00 0.12 0.03 0.05 0.07 0.00 0.03 0.24 0.32 0.09 0.09 0.13 -0.01 -0.05 0.03 0.10 0.15 0.06 0.08 0.04 0.02 0.18 -0.05 0.00 -0.01 AT5G46890 -0.06 0.03 0.13 -0.12 0.07 -0.09 0.02 0.07 0.48 -0.04 -0.01 -0.01 -0.01 -0.02 -0.10 -0.08 -0.02 -0.04 0.04 0.70 -0.03 -0.13 -0.09 -0.07 AT1G10657 0.11 0.00 0.27 0.09 0.27 0.15 -0.09 -0.10 0.13 0.12 0.16 0.25 0.33 0.29 0.26 0.23 0.23 0.12 0.03 0.02 0.26 0.35 0.16 AT1G51400 -0.05 0.14 -0.11 0.16 0.10 0.12 0.06 0.02 -0.06 0.02 -0.01 0.05 0.17 0.09 0.19 0.24 -0.05 -0.03 0.02 -0.04 0.09 0.15 AT1G13600 0.04 0.03 0.11 0.02 -0.09 0.17 -0.13 -0.08 0.14 -0.05 0.03 0.04 0.09 -0.10 -0.13 -0.15 0.13 0.15 -0.14 0.17 -0.13

Figure1.

  Figure1. Phosphorus deficiency prevents iron deficiency-induced chlorosis in evolutionarily distant plant species. A-C) Duckweed (Lemna gibba), rice (Oryza sativa cv Nipponbare), and Arabidopsis thaliana plants grown on media containing iron and phosphorus (+Fe+P), deficient in iron (-Fe+P), or deficient in both elements

Figure 2 .

 2 Figure 2. Kinetics of chlorophyll accumulation, photosystem II activity, and transcriptome change in response to iron and phosphorus availability. A-B) Chlorophyll content and PSII activity (Fv/Fm) in response to iron and/or phosphate deficiency in A. thaliana. Seedlings were grown for 7 days in the presence of iron and phosphorus (+Fe+P) and transferred to three different media: +Fe+P, -Fe+P, or -Fe-P for 15h, 28h, 39h, 52h, 76h, 100h, 124h, 148h, 172h. A) Chlorophyll data shown are from 3 experiments, each experiment with 8 plants. Error bars represent 95% confidence interval. B) Plants were dark-adapted for 30 min before measuring fluorescence kinetics from a leaf. Fv/Fm data shown are from 3 experiments, each experiment with 16 plants. Error bars represent 95% confidence interval. C) Experimental design for transcriptomic studies on A. thaliana (Col-0) shoots. Plants were grown in media containing iron and phosphorus (+Fe+P) for 7 days, transferred to three different media (+Fe+P, -Fe+P, or -Fe-P) for 39h, 52h, or 76h, and shoots were harvested for RNA extraction and sequencing. D) Global expression analysis of genes in response to -Fe+P and -Fe-P relative to +Fe+P. Numbers of genes displaying at least 2-fold change (p-value < 0.05) in their expression are shown for

Figure 3 .

 3 Figure 3. PHT4;4 prevents chlorosis under the combined deficiency of iron and phosphorus. A) A Manhattan plot for genome-wide association mapping using principal component 1 that explained 89.5% of expression variation of the 32 photosynthesis related genes across 727 A. thaliana accessions 15 . The five chromosomes are depicted by light and dark blue colors. Dashed lines correspond to FDR 5% threshold (blue) and Bonferroni α= 0.05 (red). The light grey rectangle highlights a significant association located in an intergenic region (SNP: 4493712). Two significant associations that were followed up in this study are highlighted in red rectangles. B) Representative images of wild-type Col-0, pht4;4, vtc4, and a line expressing genomic PHT4;4 in pht4;4 (pht4;4-CL) grown for 7 days in the presence of iron and phosphorus (+Fe+P) and transferred to three different media: +Fe+P, -Fe+P, or -Fe-P for 7 additional days. Scale bars: 7 mm C) Total chlorophyll content in Col-0, pht4;4, vtc4, and PHT4;4-CL grown under -Fe+P, +Fe-P, -Fe-P, or -Fe+P+AsA. Data shown are from 10 plants conducted in three independent experiments. D) mRNA abundance of VTC genes (VTC1, VTC2, and VTC4) relative to Ubiquitin 10 (At4g05320) in shoots of A. thaliana Col-0 plants grown in the presence of iron and phosphorus (+Fe+P) for 7 days and transferred to +Fe+P, -Fe+P, -Fe-P or -Fe-P+AsA for 52h. Data shown are from 3 experiments. Error bars represent 95% confidence interval. E) Total ascorbic acid (AsA) content in Col-0, pht4;4, vtc4, and PHT4;4-CL plants grown for 7 days in the presence of iron and phosphorus (+Fe+P) and transferred to +Fe+P, -Fe+P, or -Fe-P for 52h. Data shown are from 3 experiments, each with 16 plants. In box plots (C, E) center lines show sample medians; box limits indicate the 25th and 75th percentiles; whiskers extend

Figure 4 .

 4 Figure 4. bZIP58 regulates photosynthesis related genes and chlorophyll accumulation. A) mRNA abundance of 32 photosynthesis-related genes relative to Ubiquitin 10 in shoots of A. thaliana Col-0, pht4;4, and bzip58 plants grown for 7 days in the presence of iron and phosphorus (+Fe+P) and transferred to +Fe+P, -Fe+P, -Fe-P, or -Fe-P+AsA for 52h. Data were averaged from three independent experiments. Scale bar represents the relative mRNA abundance. B) mRNA abundance of bZIP58 relative to Ubiquitin 10 in A. thaliana Col-0 shoots of plants grown for 7 days on +Fe+P and transferred to +Fe+P, -Fe+P, -Fe-P, or +Fe+P+H 2 O 2 for 52h. Data are shown from 3 experiments. Error bars represent 95% confidence interval. C) VTC4 mRNA abundance relative to Ubiquitin 10 in the shoots of Col-0 and bzip58 mutants grown in the presence of +Fe+P for 7 days and transferred to +Fe+P, -Fe+P, or -Fe-P for 52h. Data shown from 3 experiments. Error bars represent 95% confidence interval. D) Confocal microscopy images of p35S::bZIP58::GFP expressing plants (scale bar = 20 µm) grown for 7 days under +Fe+P. E) Representative images of Col-0, bzip58, and a line expressing genomic bZIP58 in bzip58 mutants (bzip58-CL) grown for 7 days in +Fe+P and transferred to +Fe+P, -Fe+P, or -Fe-P for 7 additional days. Scale bars: 7 mm. F) Total chlorophyll content in Col-0, bzip58, and bZIP58-CL plants grown for 7 days in +Fe+P and transferred to -Fe+P, +Fe-P, -Fe-P, or -Fe-P+AsA for 7 days. Data shown are from 4 experiments. G) A schematic model delineating a signaling pathway that integrates Fe and P availability cues to regulate chlorophyll accumulation and photosynthesis genes. Fe deficiency (-Fe+P) causes a decrease in the expression of bZIP58 that is central to controlling the transcription of nuclear-encoded photosynthetic genes. P limitation under Fe deficiency (-Fe-P) prevents this downregulation of bZIP58 and induces VTC4. The induction of VTC4 expression requires bZIP58, whose effect could be direct or indirect, represented here by 'X'. We propose that induction of VTC4 increases ascorbic acid in the chloroplast mediated by PHT4;4. We

Figure S2 .

 S2 Figure S2. Transcriptome kinetics of A. thaliana in response to Fe and/or P deficiency. Volcano plots of individual transcript abundance in wild-type plants (Col-0) grown in -Fe+P (A,C,E) or -Fe-P (B,D,F) relative to +Fe+P. Shoot samples were collected from plants grown for 39h (A,B), 52h (C,D), or 76h (E,F). x-axis: foldchanges; y-axis: adjusted p-values based on Benjamini-Hochberg correction; Both axes use log scales. Red: log2FoldChange>|2| and -log10P>6; Blue: log2FoldChange<|2| and -log10P>6; Green: log2FoldChange>|2| and -log10P<6; Grey: log2FoldChange<|2| and -log10P<6.

Figure S3 .

 S3 Figure S3. Common and unique genes regulated by Fe and/or P deficiency. A-C) Venn diagrams show the genes that are commonly and specifically increased or decreased in abundance in shoots of A. thaliana wild type (Col-0) plants grown in

Figure S4 .

 S4 Figure S4. Gene Ontology enrichment analysis of genes specifically regulated by Fe and/or P deficiency. A, C) Heatmaps representing changes in the expression of genes that were specifically decreased in abundance under -Fe-P but not -Fe+P relative to +Fe+P (A) and increased in abundance in -Fe-P but not in -Fe+P relative to +Fe+P (C). E) A heatmap representing the expression of genes specifically upregulated by -Fe+P but not by -Fe-P relative to +Fe+P. B, D, F) Gene Ontology enrichment for biological processes (GO-BP) in the genes that were specifically decreased by -Fe-P relative to +Fe+P (B), specifically increased by -Fe-P relative to +Fe+P (D), and specifically increased by -Fe+P relative to +Fe+P (F) using GENEMANIA22 . Number of genes in each functional category and adjusted p-values for the enrichment are shown. FDR = false discovery rate.

Figure S5 .

 S5 Figure S5. Correlation in the expression of the 32 photosynthesis genes specifically downregulated by -Fe+P across 727 A. thaliana accessions. A) Aheatmap of pairwise correlations (Pearson's correlation coefficient) in the 32 genes across 727 Arabidopsis accessions grown under control conditions. The correlations were calculated using normalized read counts. B) Principal Component (PC) Analysis was performed using the expression of 32 genes in 727 Arabidopsis accessions. Xand Y-axes show PC 1 and PC 2 that explain 89.5% and 5.9 % of the total variance, respectively.

Figure S6. A close-up view of chromosomes 1 and 4 around bZIP58 and PHT4; 4

 4 Figure S6. A close-up view of chromosomes 1 and 4 around bZIP58 and PHT4;4 respectively. A Manhattan plot for genome-wide association mapping using PC 1 of the expression profile of the 32 photosynthesis genes across 727 accessions. The five A. thaliana chromosomes are depicted by light and dark blue colors. Blue and red horizontal dashed lines correspond to FDR 5% threshold and

Figure S7 .(

 S7 Figure S7. Effects of Fe and/or P availability on chlorophyll content in A. thaliana Col-0 and mutants of candidate genes identified using GWAS. Chlorophyll content in Col-0 (CS60000), T-DNA insertion mutant lines in AT1G13570 (SALK_139877), AT1G13580 (SALK_150849), AT1G13590 (SALK_063177), AT1G13605 (SALK_087271), AT1G13607 (SALK_130208), AT1G13608 (SALK_023173), AT1G13609 (SAIL_1243_E04), AT1G13610 (SAIL_897_D11), At4g00355 (N469134), AT4G00360 (SALK_128714), AT4G00380 (SAIL_842_E09), AT4G03585 (SALK_ 128714), At4g00390 (SAIL_313_F07), and At4g00400 (SAIL_633_E10) grown for 7 days in the presence of iron and phosphorus (+Fe+P) and transferred to +Fe+P, -Fe+P, or -Fe-P for an additional week. Data shown from 3 experiments. Error bars represent 95% confidence interval. Letters a and b indicate significantly different values at p <0.05 determined by one-way ANOVA and Tukey's honest significant difference (HSD) tests. Source data are provided as a Source Data file.

  Figure 1

  Figure 3

FDR

  Cellular respira6onEnergy deriva6on by oxida6on of organic compounds Oxida6on-reduc6on process Genera6on of precursor metabolites and energy

  preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21 Shaikhali, J. & Wingsle, G. Redox-regulated transcription in plants: emerging concepts. AIMS Mol. Sci. 4, 301 (2017). 22 Warde-Farley, D. et al. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 38, W214-W220 (2010). 23 Haydon, M. J. et al. Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis.

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M. & Ainsworth, E. A. Measurement of reduced, oxidized and total ascorbate content in plants. Nat. Protoc. 2, 871-874 (2007). 28 Alexieva, V., Sergiev, I., Mapelli, S. & Karanov, E. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ. 24, 1337-1344 (2001). 29 Gechev, T., Mehterov, N., Denev, I. & Hille, J. A simple and powerful approach for isolation of Arabidopsis mutants with increased tolerance to H2O2-induced cell death. Methods Enzymol. 527, 203-220 (2013). 30 Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357-360 (2015). 31 Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537-W544 (2018). 32 Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). 33 Rouached, H. et al. Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1. 1 and SULTR1. 2, in Arabidopsis. Plant Physiol. 147, 897-911 (2008). 34 Consortium, O. T. O. G. 1,135 genomes reveal the global pattern of polymorphism in Arabidopsis thaliana. Cell 166, 481-491 (2016). 35 Seren, Ü. et al. GWAPP: a web application for genome-wide association mapping in Arabidopsis. Plant Cell 24, 4793-4805 (2012). 36 Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57, 289-300 (1995). 37 Spitzer, M., Wildenhain, J., Rappsilber, J. & Tyers, M. BoxPlotR: a web tool for generation of box plots. Nat. Methods 11, 121 (2014).

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	. bZIP58
	complemented lines (bZIP58-CL) were generated by expressing 3896 bp genomic
	DNA containing bZIP58 in the bzip58 mutant background (NASC, N571881).
	Complementation of pht4;4 mutant plants (PHT4;4-CL) was obtained by expressing
	6450 bp genomic DNA containing PHT4;4 in the pht4;4 mutant background (NASC,
	without sucrose supplementation, and 0.8% washed agar. The agar was washed 3
	times with 50 mM EDTA, pH 5.7, with continuous stirring for 16 h, then washed 6
	times with Milli-Q de-ionized water for 2 hours to reduce mineral contamination 23 . P-
	deficient media contained 12.49 µM KH 2 PO 4 (+Fe-P). Fe-free media was obtained by
	omitting FeSO 4 .7H 2 O from the growth media (-Fe+P). P-and Fe-deficient media
	contained 12.49 µM KH 2 PO 4 (+Fe-P), and no FeSO 4 .7H 2 O (-Fe-P). Seeds were
	stratified at 4°C for 3 days and grown on vertical agar plates in a growth chamber
	with 22 °C, 24 h of light at 100 µmol m -2 s -1 fluorescent illumination. Lemna gibba
	(duckweed) plants used during this study were obtained from Duckweeds stock
	center (stock number 29-DWC131) at Rutgers University (USA). Duckweed plants
	were grown in 1X Schenk & Hildebrandt (SH) hydroponic medium containing 0.05%
	2-(N-morpholino)ethanesulfonic acid (MES) and 1% sucrose, and pH adjusted to 5.7.
	For experiments with duckweeds, P-deficient and Fe-deficient media contained 1%
	NH 4 H 2 PO4 and 1% FeSO 4 7H

N469134

). Arabidopsis plants were grown on control (+Fe+P) plates containing 1.249 mM KH 2 PO 4 ; 0.25 mM Ca(NO 3 ) 2 ; 0.5 mM KNO 3 ; 1 mM MgSO 4 ; 100 μM FeSO 4 .7H 2 O; 30 μM H 3 BO 3 ; 1 μM ZnCl 2 ; 10 μM MnCl 2 ; 1 μM CuCl 2 ; 0.1 μM (NH 4 ) 6 Mo 7 O 24 ; and 50 μM KCl; 0.05% 2-(N-morpholino)ethanesulfonic acid (MES), 2 O, respectively, of 1X SH media. Media were changed every 7 days. The growth condition was 22 °C and 24 h of light at 80 µmol m -2 s -1 .

preprint

  AT3G46780-0.14 0.85 0.00 0.20 -0.03 -0.19 -0.13 0.21 0.06 0.30 0.72 0.30 0.78 0.67 -0.27 -0.14 0.04 0.09 0.40 0.82 AT5G47550 -0.08 -0.09 -0.02 -0.03 0.06 0.05 0.08 0.14 0.03 -0.16 -0.05 -0.05 -0.03 0.17 0.10 0.11 0.03 0.03 -0.09 AT3G21055 -0.09 0.21 -0.06 -0.20 -0.25 0.12 -0.05 0.28 0.62 0.29 0.74 0.63 -0.32 -0.13 0.03 0.02 0.32 0.69 AT5G59780 -0.09 0.10 0.26 0.09 0.20 0.20 0.01 0.11 0.25 0.00 0.07 0.17 0.01 0.06 0.12 0.23 -0.04 AT1G06980 0.16 0.06 0.09 -0.01 -0.20 -0.09 0.19 0.05 0.35 0.29 -0.02 0.01 0.00 -0.01 -0.23 0.27 AT1G80340 -0.16 -0.08 -0.03 -0.05 -0.01 -0.01 -0.06 0.09 0.06 0.02 0.42 0.05 -0.17 -0.07 -0.01 AT5G06510 0.30 0.18 0.08 -0.02 -0.10 0.26 -0.09 -0.03 0.28 -0.07 -0.05 0.42 -0.01 -0.11 AT5G51710 0.27 0.19 -0.10 -0.09 0.08 -0.07 -0.13 0.26 -0.05 -0.07 0.35 -0.09 -0.09

	AT1G76800	0.44 -0.02 0.19 0.32 0.23 0.18 0.09 -0.03 0.15 0.34 0.29 0.21
	AT5G63850	-0.03 0.01 0.23 0.13 0.08 0.24 0.01 0.13 0.38 0.30 0.08
	AT5G08030	0.34 0.16 0.19 0.27 -0.13 -0.01 -0.04 0.02 0.35 0.22
	AT5G39530	0.26 0.60 0.60 -0.28 -0.14 -0.01 0.01 0.45 0.65
	AT4G01070

0.17 0.15 -0.11 -0.12 0.15 0.29 0.51 0.20 AT3G61470 0.81 -0.06 -0.05 0.01 0.17 0.20 0.87 AT2G30570 -0.09 -0.06 0.04 0.09 0.30 0.72 AT4G04955 0.12 0.05 0.24 -0.17 -0.17

Acknowledgements

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The authors thank the members of Rhee lab; Benoit Lacombe and HONUDE team (INRAe) for their comments on the manuscript and helpful discussions. We thank the ICP-MS/TIMS Facility within Stanford University for assistance with the ICP-MS measurements, and the Stanford Functional Genomics Facility for assistance with RNA sequencing (Stanford, CA). The GWAS analysis was made possible by data generated by Kawakatsu et al. 15 .

Funding

This work was funded in part by the "Institut National de la Recherche Agronomique -Montpellier -France" INRA, the AgreeenSkills Plus, and Michigan State University (USA) to H.R. as well as by the Carnegie Institution for Science, Brigitte Berthelemot, National Science Foundation (IOS-1546838, IOS-1026003), and the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program grant nos. DE-SC0018277, DE-SC0008769, and DE-SC0020366 to S.Y.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

RNA-seq experiments

Arabidopsis wild type (Col-0) plants were grown in control (+Fe+P) media for 7 days and transferred to three different media: control (+Fe+P), iron deficiency (-Fe+P), and iron and phosphate deficiency (-Fe-P) conditions. Shoots were collected at 39h, 52h and 76h after the transfer. For RNA-seq experiments, three biological replicates were prepared for each time point (39h, 52h and 76h) and each condition (+Fe+P, -Fe+P and -Fe-P) for a total of 27 samples. Total RNA was extracted from these samples using RNeasy Plant Mini Kit (QIAGEN) using the RLT buffer supplemented with 2mercaptoethanol, and RNA quality was verified using an Agilent 2100 BioAnalyzer.

The mRNAs were subsequently isolated using magnetic KAPA Biosystems oligo-dT beads from KAPA Biosystems (Roche) and then used for library construction using the KAPA Biosystems RNA HyperPrep Kit (Roche). To index the libraries, we used adapters from the KAPA Biosystems Single-Indexed Adapter Set A+B (Roche).

Before pooling the libraries, we monitored their quality and concentrations using an Agilent 2100 BioAnalyzer, Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the KAPA Library Quantification Kit (Roche). Pooled libraries were then sequenced using the NextSeq 500 System at the Stanford Functional Genomics Facility (Stanford, CA). Raw reads were demultiplexed and aligned to the TAIR10 genome annotation using HISAT2 30 on the Galaxy web platform 31 . Finally, mapped read counts were used to perform normalization and a differential expression analysis on R using the DESeq2 32 and TxDB.Athaliana.BioMart.plantsmart 28 (Bioconductor) packages. In DESeq2, p-values from the Wald test were corrected for multiple hypothesis testing using the Benjamini and Hochberg method. A transcript was considered differentially expressed if the adjusted p-value < 0.05. Volcano plots were generated using the EnhancedVolcano package (version 1.6.0) (Bioconductor) with a default cut-off of log2(Fold Change) > |2| and adjusted p value < 10e -6 . DEGs having a p-value of 0 were converted to 10 -1 x lowest non-zero p-value.

Real-time quantitative reverse-transcription PCR

Seeds of Arabidopsis wild type (Col-0), bzip58, and pht4;4 mutant plants were germinated and grown for 7 days in control (+Fe+P) media, and then transferred to preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Competing interests

No competing interests declared. Data supporting the findings of this work are available within the paper and its Supplementary Information and Source Data files. The datasets and plant materials generated and analyzed during the current study are available from the corresponding author H.R. upon request. Transcriptome data were deposited in NCBI's Gene Expression Omnibus (GEO) under a project number GSE163190.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.