**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** URGENT ACTION IS NEEDED FOR SUSTAINABLEINCREASES IN WHEAT PRODUCTIVITY TO FEED A GROWING HUMAN POPULATION. INCREASES IN GRAIN YIELD REQUIRE SIMULTANEOUS INCREASES IN SINK TRAITS (E.G. GRAIN SIZE AND NUMBER) AND SOURCE TRAITS (E.G. BIOMASS, PHOTOSYNTHESIS). SEVERAL GENES INCREASING GRAIN SIZE AND NUMBER HAVE BEEN IDENTIFIED RECENTLY, BUT GENES CONTROLLING SOURCE TRAITS REMAIN LESS EXPLORED. MOST OF THE RESOURCES USED TO FILL GRAINS DURING REPRODUCTIVE DEVELOPMENT ARE GENERATED EARLIER DURING VEGETATIVE DEVELOPMENT. THEREFORE, ENHANCING EARLY GROWTH AND BIOMASS ACCUMULATION CAN COMPLEMENT IMPROVEMENTS ON 'SINK' TRAITS.THE VEGETATIVE STAGE HAS BEEN DIVIDED IN TWO MAIN PHASES, JUVENILE AND ADULT, WITH THE TRANSITION BETWEEN THESE TWO PHASES (REFERRED AS VEGETATIVE PHASE CHANGE) ASSOCIATED TO THE ACQUISITION OF COMPETENCE TO FLOWER, TO CHANGES IN LEAF MORPHOLOGY, GROWTH RATE AND PLANT MORPHOLOGY. THE DURATION OF THESE PHASES HAS A STRONG IMPACT IN PLANT PRODUCTIVITY BY AFFECTING THE SUCCESSFUL ESTABLISHMENT OF THE PLANTS IN THE SOIL, REGULATING THE ARCHITECTURE OF THE PLANT TO MAXIMIZE ENERGY HARVESTING, AND DETERMINING THE AMOUNT OF BIOMASS ACCUMULATED THAT CAN BE USED LATER FOR GRAIN FILLING.THE JUVENILE PHASE IS CHARACTERIZED BY SMALLER AND STRESS TOLERANT ORGANS, WHICH ARE IMPORTANT FOR SEEDLINGESTABLISHMENT IN ENVIRONMENTS OF LIMITED RESOURCES, BUT THAT ARE UNNECESSARY UNDER OPTIMAL CULTIVATION CONDITIONS. WE HYPOTHESIZE THAT SHORTENING OR BYPASSING THE CONSERVATIVE JUVENILE PHASE WILL BOOST EARLY GROWTH AND BIOMASS ACCUMULATION AND HAVE A POSITIVE IMPACT ON GRAIN YIELD IN FAVORABLE ENVIRONMENTS. THEREFORE, THE AIM OF THIS PROPOSAL IS TO INCREASE WHEAT PRODUCTIVITY BY MANIPULATING THE LENGTH OF THE VEGETATIVE JUVENILE PHASE.THE JUVENILE-ADULT PHASE TRANSITION IS CONTROLLED BY THE LEVELS OF AN ENDOGENOUS SMALL MOLECULE CALLED MICRORNA MIR156. MICRORNAS (MIRNAS) ARE ENDOGENOUS, SMALL NON-CODING RNAS THAT FUNCTION IN POST-TRANSCRIPTIONAL REGULATION OF GENE EXPRESSION. MIR156 EXERTS ITS FUNCTION BY RECOGNIZING A COMPLEMENTARY SEQUENCE IN A GROUP OF GENES OF THE SPL TRANSCRIPTION FACTOR FAMILY AND REPRESSING THEIR EXPRESSION.IN SEVERAL PLANT SPECIES MIR156 EXPRESSION IS HIGHER IN JUVENILE ROOTS AND SHOOTS, INCLUDING JUVENILE LEAVES,AND THEN DECLINES WITH PLANT AGE WITH A PARTICULAR ABRUPT CHANGE DURING VEGETATIVE PHASE CHANGE. IF THE EXPRESSION OF MIR156 IS MAINTAINED CONSTITUTIVELY HIGH THE JUVENILE PHASE IS PROLONGED, WHILE REDUCTION OF MIR156 ACTIVITY ELIMINATES JUVENILE TRAITS ACCELERATING THE TRANSITION TO ADULT PHASE. THESE OBSERVATIONS INDICATED THAT MIR156 IS BOTH NECESSARY AND SUFFICIENT FOR THE JUVENILE PHASE.IN WHEAT WE IDENTIFIED 5 GENES PER GENOME THAT ENCODE MIR156 PRECURSOR SEQUENCES THAT CAN BE POTENTIALLY PROCESSED TO GENERATE ONE OR MORE MATURE MIR156S. IN ADDITION, WE IDENTIFIED 18 SPL GENES, WITH NINE LIKELY TARGETED BY MIR156. ANALYSIS OF GENE EXPRESSION REVEALED THAT AMONG THESE NINE MIR156-TARGETED SPL GENES, SPL3AND SPL4 HAVE THE HIGHEST EXPRESSION IN VEGETATIVE TISSUES, WITH SPL13 AND SPL23 ALSO BEING EXPRESSED BUT AT A LOWER LEVEL. SPL3, SPL4 AND SPL23 ARE THE ONLY MIRNA156-TARGETED SPLS THAT ARE ALSO EXPRESSED IN ROOTS. SINCE OUR PROJECT IS FOCUSED ON IMPROVING EARLY GROWTH, WE JUST FOCUS ON THESE FOUR SPLS.WE PREVIOUSLY STUDIED THE ROLE OF THE MIR156/SPL MODULE IN WHEAT BY TRANSGENIC APPROACHES. WE GENERATED TRANSGENIC LINES THAT EXPRESSES CONSTITUTIVE HIGH LEVELS OF MATURE MIR156 (UBI::MIR156) AND OTHERS THAT EXPRESSED AN ARTIFICIAL MIR156 TARGET THAT BINDS AND SEQUESTERS MIR156, RESULTING IN REDUCED MIR156 ACTIVITY. THIS LAST APPROACH IS CALLED ARTIFICIAL TARGET MIMICRY APPROACH AND IS REFERRED HEREAFTER TO AS MIM156.ADULT UBI::MIR156 PLANTS WITH CONSTITUTIVE EXPRESSION OF MIR156 PRODUCED SMALLER LEAVES, SHORTER ROOTS, MORE TILLERS AND REDUCED LEVELS OF SPL3, SPL4, SPL13 AND SPL23, ALL FEATURES TYPICALLY OBSERVED IN JUVENILE PLANTS. THESE PLANTS HAVE REDUCED FERTILITY.CONVERSELY, MIM156SEEDLINGS HAD ADULT CHARACTERS, INCLUDING LARGER LEAVES, LONGER ROOTS, LESS BUT MORE ROBUST TILLERS WITH THICKER STEMS AND HIGHER EXPRESSION OF SPL3, SPL4, SPL13 AND SPL23 IN THE FIRST LEAF. THESE PLANTS HAVE NORMAL FERTILITY AND GRAIN PRODUCTION. TAKEN TOGETHER, THESE PRELIMINARY RESULTS SUGGESTED THAT MIR156 ACTIVITY LIMITS ROOT AND SHOOT GROWTH DURING THE FIRST WEEKS OF DEVELOPMENT, LIKELY BY REPRESSING SPL GENES. THIS OBSERVATION RAISES THE EXCITING POSSIBILITY THAT A PRECISE MANIPULATION OF MIR156 AND/OR SPL ACTIVITY DURING THE WHEAT JUVENILE PHASE MAY RESULT IN EARLY GROWTH VIGOR AND DEEPER ROOTS. IN ADDITION, THE LESS BUT MORE ROBUST TILLERS OBSERVED IN MIM156 LINES SUGGEST THAT REDUCED MIR156 DOSAGE COULD ALSO RESULT IN AN IMPROVED WHEAT PLANT ARCHITECTURE.WE USED AN EFFICIENT TRANSFORMATION AND CRISPR EDITING SYSTEM FOR WHEAT RECENTLY DEVELOPED IN OUR LABORATORY TO SUCCESSFULLY TARGET ALL MIR156 GENES USING FIVE GUIDE RNAS. IN ADDITION, WE INCREASED THE EXPRESSION OF THE SELECTED SPL GENES BY INTRODUCING POINT MUTATIONS IN THEIR MIR156 BINDING SITES. THESE DOMINANT SPL MUTANTS ARE DESIGNATED AS RESISTANT SPLS OR RSPLS. WE HAVE ALREADY IDENTIFIED RSPL3, RSPL4, AND RSPL23 IN OUR SEQUENCED MUTANT POPULATIONS, AND WILL GENERATE RSPL13 MUTANTS USING CRISPR-CAS9.BASED ON THE PRELIMINARY DATA FROM OUR TRANSGENIC EXPERIMENTS AND THE AVAILABLE GENETIC MATERIALS, THE GENERAL OBJECTIVE OF THIS GRANT IS TO MANIPULATE THE DURATION OF THE EARLY STAGES OF VEGETATIVE DEVELOPMENT IN TETRAPLOID WHEAT TO INCREASE GRAIN YIELD POTENTIAL BY INCREASING THE INITIAL ACCUMULATION OF BIOMASS PER TILLER AND OPTIMIZING PLANT ARCHITECTURE. THE SPECIFIC OBJECTIVES ARE TO: I) MANIPULATE MIR156 DOSAGE BY COMBINING AVAILABLE CRISPR-INDUCED MUTATIONS IN FIVE MIR156 GENES IN BOTH A AND B TETRAPLOID WHEAT GENOMES. II) MANIPULATE SPL3, SPL4, SPL13, AND SPL23 EXPRESSION BY GENERATING MUTATION IN THEIR MIR156 BINDING SITES. III) QUANTIFY THE EFFECT OF DIFFERENT MUTANT COMBINATIONS ON ROOT AND SHOOT GROWTH IN GREENHOUSE AND HYDROPONIC EXPERIMENTS AND THEIR EFFECT ON GRAIN YIELD IN FIELD EXPERIMENTS. WE PLAN TO CREATE A BROAD RANGE OF TRANSGENE-FREE MUTANTS WITH ALTERED DOSAGE OR SPATIO-TEMPORAL EXPRESSION PATTERN OF ENDOGENOUS MIR156 OR SPL GENES. WE HYPOTHESIZE THAT THESE LINES WILL HAVE ENHANCED GROWTH DURING THE JUVENILE PHASE WITH LIMITED NEGATIVE PLEOTROPIC EFFECTS. THESE GENETIC STOCKS WILL BE PUBLICLY AVAILABLE TO WHEAT BREEDERS AND RESEARCHERS. THE ULTIMATE GOAL IS TO GENERATE NOVEL GENETIC VARIATION THAT OPTIMIZE THE DURATION OF THE WHEAT JUVENILE PHASE AND MAXIMIZE BIOMASS PRODUCTION PER TILLER TO SUPPORT INCREASE GRAIN YIELD WHEN COMBINED WITH FAVORABLE ALLELES FOR SINK TRAITS.