Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.06.29.662209

An ARF2-GRF5 module, operating early and upstream of GLK factors, reveals genetic hierarchy during chloroplast biogenesis

Mishra, P.·Hibberd, J. M.·Lopez-Juez, E.
View original source ↗Export metadata ↓

Abstract

O_LIOur current understanding of the regulation of chloroplast development lags behind that of the process itself. The GOLDEN2-LIKE (GLK) factors drive the expression of photosynthesis-associated nuclear (PhAN) genes. Simultaneous loss of GLK1 and GLK2 in Arabidopsis plants leads to pale-green, incompletely-developed chloroplasts. This highlights the GLKs role but also the involvement of other components. C_LIO_LIWe hypothesised that further regulators could be found by searching for suppressor mutations of a severe glk1 knock-down (KD) glk2 knock-out (KO) mutant. We identified suppressor of glks (sgl) 2, sgl2. C_LIO_LIThe sgl2 glk1 glk2 triple mutant showed significant increases in chlorophyll, expression of PhAN genes, chloroplast thylakoid stacking, and cellular volume of chloroplasts. sgl2 was caused by loss of function of AUXIN RESPONSE FACTOR2, ARF2. arf2sgl2 boosted chloroplast content as a single mutant, and dramatically suppressed the chloroplast delay of the phytochrome-deficient long hypocotyl 1 (hy1) mutant. Overexpression of Growth Regulating Factor 5 (GRF5), which is negatively regulated by ARF2, also rescued chlorophyll levels in glk1 glk2, confirming a role for ARF2 and GRF5 as a transcription module. We observed in glk1 glk2 low but detectable expression of GLK1, which was elevated in arf2sgl2 glk1 glk2, and saw reduced suppression by arf2sgl2 of the phenotype of a separate glk1-2 glk2-2 mutant carrying two KO alleles, revealing partial epistasis. Presumed orthologs of ARF2 and GRF5 are expressed very early in developing cereal leaves, well ahead of the bulk of GLK expression during the greening stage. C_LIO_LIOur results place ARF2 as a negative switch modulating GRF5 early and upstream, whether directly or indirectly, of GLK factors, but also acting independently from them. This reveals a hierarchy of transcription factor action and feed-forward regulation in chloroplast biogenesis during leaf development. C_LI

Explore related subjects

plant biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Mishra, P., Hibberd, J. M., Lopez-Juez, E.. 2025-07-01. An ARF2-GRF5 module, operating early and upstream of GLK factors, reveals genetic hierarchy during chloroplast biogenesis. https://doi.org/10.1101/2025.06.29.662209

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Seeding paradise: germination ecophysiology of Xyris paradisiaca Wand. (Xyridaceae), an endangered endemic species from Central Brazil

Seed ecophysiology is essential for understanding plant regeneration and developing effective conservation and ecological restoration strategies, yet knowledge remains scarce for most threatened Cerrado species. We characterised the germination niche, desiccation tolerance and storage behaviour of Xyris paradisiaca (Xyridaceae), an Endangered species endemic to Central Brazil, to assess whether its restricted distribution is associated with narrow germination requirements and to inform seed-based conservation. Germination was tested across light regimes, constant temperatures, decreasing water potentials and short-duration heat shocks, while thermal- and hydro-time models were used to quantify thermal and hydric thresholds. Seeds exhibited an absolute light requirement but broad abiotic tolerances. Germination remained >87% between 15 and 40 {degrees}C, with estimated cardinal temperatures of Tb = 8.94 {degrees}C, To = 33.13 {degrees}C and Tc = 45.53 {degrees}C. Germination remained similar to the control down to -0.6 MPa and exceeded 40% at -1.0 MPa, with a median base water potential of {Psi}b = -1.03 MPa. Germination also remained high after 1-min heat shocks up to 200 {degrees}C. Seeds were desiccation tolerant, with 100% germination after drying and a viability loss index of -0.04, and germination remained >90% after 24 months of ambient storage. Thus, the highly restricted distribution of X. paradisiaca is not driven by a narrow physiological germination niche. Its broad environmental tolerances, desiccation tolerance and favourable storage behaviour also highlight its unexplored potential for seed-based restoration, propagation and ex situ conservation.

plant biology↗
bioRxiv2026

Planted origin of shade trees: a robust determinant of cocoa yield among smallholder farmers in Cote d'Ivoire

CONTEXT: While the agroeconomic literature on the determinants of cocoa yield among West African smallholders is abundant, it remains dominated by descriptive studies or work conducted at an aggregated regional scale, leaving open the question of the role of shade-tree management practices at the scale of the individual plantation. OBJECTIVE: This study tests whether shade-tree management practices and the farmer's socio-demographic profile explain variation in cocoa yield, using a nationwide sample of 409 plantations covering, for the first time, the three major Ivorian production zones (loops) (98, 151 and 160 plantations for loops 1, 2 and 3, respectively). METHODS: A univariate screening of 27 variables, a mixed model with a random intercept by village, a production-function specification, LASSO variable selection, and a random forest were applied, all evaluated by 5-fold cross-validation. RESULTS AND CONCLUSIONS: The five approaches converge on a robust result: the deliberately planted origin of shade trees (as opposed to a residual or spontaneous origin) is the strongest and most stable determinant of yield, with a mean gap of 332 versus 183 kg/ha/year. This effect withstands four successive robustness checks: it remains significant after simultaneous adjustment for age, plantation size, production zone, technical extension, and farmer education; it is not driven by a handful of extreme plantations; it holds within each of the three production zones taken separately rather than in only one of them; and, taken in isolation, it retains a positive out-of-sample predictive power (cross-validated R2=8776; 0.05). A second group of robust determinants of more modest magnitude emerges for agricultural technical extension (ANADER/CNRA/SATMACI) and farmer education level; shade-tree alignment shows a signal in the same direction, consistent with recent independent work in Cote d'Ivoire, but becomes statistically marginal once adjusted for these other factors. The full multivariate model reaches a cross-validated R2 of around 0.07-0.08. SIGNIFICANCE: This signal, undetectable in an analysis restricted to loop 1 alone (n=98) for lack of statistical power, confirms the value of nationwide sampling for detecting modest but real agronomic determinants, and argues for integrating shade-tree origin into agroforestry extension programmes.

plant biology↗
bioRxiv2026

Parental Genome Assemblies of Suyunuo1 Reveal Structural Variation Underlying Edible Waxy Maize Evolution, Superior Hybrid Performance and Yield - Flavor Balance

Edible waxy maize is a unique domesticated cereal valued for its superior sensory and nutritional properties, yet high quality gap free genomes remain lacking, hindering the exploration of structural variations (SVs) and domestication related divergence. Here, we assembled two chromosome level, gap free genomes of the elite waxy maize inbred lines Tongxi 5 and Hengbai 522, the parents of the widely cultivated hybrid Suyunuo 1. Comparative genomic analysis revealed substantial genome size variation, prominent megabase scale SVs, and extensive knob repeat expansion in Tongxi 5. We identified widespread gene presence absence variation and hyperdivergent regions enriched in transposable elements, which largely underpin genomic differentiation between waxy and field maize. Population genomic analyses further demonstrated asymmetric introgression from field maize into the two parents, whereas the conserved waxy haplotype supports a shared ancestral origin. Integrative GWAS based on SNPs, InDels and SVs revealed that SVs substantially contribute to phenotypic diversity and mediate the co-regulation of yield traits and flavor-related metabolites. Our results uncover key genomic events underlying post-domestication divergence and highlight the essential roles of SVs in heterosis and flavor-yield balance, providing valuable genomic resources for waxy maize improvement.

plant biology↗