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SUMMARY: Symbiotic nitrogen fixation is an energy‐intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR‐Cas9‐mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate‐tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate‐triggered inhibition of nodulation, as the expression of nitrate‐responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O‐arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4‐GmNIC1a/b module in mediating high nitrate‐induced inhibition of nodulation. Significance Statement: This study highlights the crucial role of the GmNLP1/4‐GmNIC1a/b module in mediating high nitrate‐induced inhibition of nodulation. [ABSTRACT FROM AUTHOR]

Summary: Legume nodulation requires light perception by plant shoots and precise long‐distance communication between shoot and root. Recent studies have revealed that TGACG‐motif binding factors (GmSTFs) integrate light signals to promote root nodulation; however, the regulatory mechanisms underlying nodule formation in changing light conditions remain elusive.Here, we applied genetic engineering, metabolite measurement, and transcriptional analysis to study soybean (Glycine max) nodules.We clarify a fine‐tuning mechanism in response to ultraviolet B (UV‐B) irradiation and rhizobia infection, involving GmUVR8‐dependent UV‐B perception and GmSTF3/4‐GmMYB12‐GmCHS‐mediated (iso)flavonoid biosynthesis for soybean nodule formation. GmUVR8 receptor‐perceived UV‐B signal triggered R2R3‐MYB transcription factors GmMYB12‐dependent flavonoid biosynthesis separately in shoot and root. In shoot, UV‐B‐triggered flavonoid biosynthesis relied on GmUVR8a, b, c receptor‐dependent activation of GmMYB12L‐GmCHS8 (chalcone synthase) module. In root, UV‐B signaling distinctly promotes the accumulation of the isoflavones, daidzein, and its derivative coumestrol, via GmMYB12B2‐GmCHS9 module, resulting in hypernodulation. The mobile transcription factors, GmSTF3/4, bind to cis‐regulatory elements in the GmMYB12L, GmMYB12B2, and GmCHS9 promoters, to coordinate UV‐B light perception in shoot and (iso)flavonoid biosynthesis in root.Our findings establish a novel shoot‐to‐root communication module involved in soybean nodulation and reveal an adaptive strategy employed by soybean roots in response to UV‐B light. [ABSTRACT FROM AUTHOR]

Summary: The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops. [ABSTRACT FROM AUTHOR]

Summary: Phosphoglycerate kinase (PGK) is a highly conserved reversible enzyme that participates in both glycolysis and photosynthesis. In Arabidopsis thaliana, one cytosolic PGK (PGKc) and two plastidial PGKs (PGKp) are known. It remains debatable whether the two PGKp isozymes are functionally redundant or specialized in plastidial carbon metabolism and fixation. Here, using a pooled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) strategy, we found that plants with single mutations in pgkp1 or pgkp2 were not significantly affected, whereas a pgkp1pgkp2 double mutation was lethal due to retarded carbon fixation, suggesting that PGKp isozymes play redundant functional roles. Metabolomic analysis demonstrated that the sugar‐deficient pgkp1pgkp2 double mutation was partially complemented by exogenous sugar, although respiration intermediates were not rescued. Chloroplast development was defective in pgkp1pgkp2, due to a deficiency in glycolysis‐dependent galactoglycerolipid biosynthesis. Ectopic expression of a plastid targeting PGKc did not reverse the pgkp1pgkp2 double‐mutant phenotypes. Therefore, PGKp1 and PGKp2 play redundant roles in carbon fixation and metabolism, whereas the molecular function of PGKc is more divergent. Our study demonstrated the functional conservation and divergence of glycolytic enzymes. Significance Statement: There have been debates on whether different plastidial PGK isoforms play specialized or redundant roles in plastidial glycolysis and the Calvin cycle. Utilizing CRISPR/Cas9 and biochemical, metabolomic and lipidomic approaches, we found that Arabidopsis PGKp isozymes serve redundant, but essential, functions in carbon fixation, metabolism and galactoglycerolipids biosynthesis in chloroplasts. [ABSTRACT FROM AUTHOR]

Plant male gametogenesis involves complex and dynamic changes in gene expression. At present, little is known about the transcription factors involved in this process and how their activities are regulated. Here, we show that a pollen-specific transcription factor, WRKY34, and its close homolog, WRKY2, are required for male gametogenesis in Arabidopsis thaliana. When overexpressed using LAT52, a strong pollen-specific promoter, epitope-tagged WRKY34 is temporally phosphorylated by MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs, or MPKs), at early stages in pollen development. During pollen maturation, WRKY34 is dephosphorylated and degraded. Native promoter-driven WRKY34-YFP fusion also follows the same expression pattern at the protein level. WRKY34 functions redundantly with WRKY2 in pollen development, germination, and pollen tube growth. Loss of MPK3/MPK6 phosphorylation sites in WRKY34 compromises the function of WRKY34 in vivo. Epistasis interaction analysis confirmed that MPK6 belongs to the same genetic pathway of WRKY34 and WRKY2. Our study demonstrates the importance of temporal post-translational regulation of WRKY transcription factors in the control of developmental phase transitions in plants. [ABSTRACT FROM AUTHOR]