Progress and challenges in applying CRISPR/Cas techniques to the genome editing of trees

With the advent of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated protein(Cas)system,plant genome editing has entered a new era of robust and precise editing for any genes of interest.The development of various CRISPR/Cas toolkits has enabled new genome editing outcomes that not only target indel mutations but also enable base editing and prime editing.The application of the CRISPR/Cas toolkits has rapidly advanced breeding and crop improvement of economically important species.CRISPR/Cas toolkits have also been applied to a wide variety of tree species,including apple,bamboo,Cannabaceae,cassava,citrus,cacao tree,coffee tree,grapevine,kiwifruit,pear,pomegranate,poplar,ratanjoyt,and rubber tree.The application of editing to these species has resulted in significant discoveries related to critical genes associated with growth,secondary metabolism,and stress and disease resistance.However,most studies on tree species have involved only preliminary optimization of editing techniques,and a more in-depth study of editing techniques for CRISPR/Cas-based editing of tree species has the potential to rapidly accelerate tree breeding and trait improvements.Moreover,tree genome editing still relies mostly on Cas9-based indel mutation and Agrobacterium-mediated stable transformation.Transient transformation for transgene-free genome editing is preferred,but it typically has very low efficiency in tree species,substantially limiting its potential utility.In this work,we summarize the current status of tree genome editing practices using the CRISPR/Cas system and discuss limitations that impede the efficient application of CRISPR/Cas toolkits for tree genome editing,as well as future prospects.

PuHox52 promotes coordinated uptake of nitrate,phosphate, and iron under nitrogen deficiency in Populus ussuriensis

It is of great importance to better understand how trees regulate nitrogen(N) uptake under N deficiency conditions which severely challenge afforestation practices, yet the underlying molecular mechanisms have not been well elucidated. Here,we functionally characterized PuHox52, a Populus ussuriensis HD-ZIP transcription factor, whose overexpression greatly enhanced nutrient uptake and plant growth under N deficiency. We first conducted an RNA sequencing experiment to obtain root transcriptome using PuHox52-overexpression lines of P. ussuriensis under low N treatment. We then performed multiple genetic and phenotypic analyses to identify key target genes of PuHox52 and validated how they acted against N deficiency under PuHox52 regulation.PuHox52 was specifically induced in roots by N deficiency, and overexpression of PuHox52promoted N uptake, plant growth, and root development. We demonstrated that several nitrate-responsive genes(PuNRT1.1, PuNRT2.4,PuCLC-b, PuNIA2, PuNIR1, and PuNLP1),phosphate-responsive genes(PuPHL1A and PuPHL1B), and an iron transporter gene(PuIRT1) were substantiated to be direct targets of PuHox52. Among them, PuNRT1.1, PuPHL1A/B, and PuIRT1 were upregulated to relatively higher levels during PuHox52-mediated responses against N deficiency in PuHox52-overexpression lines compared to WT. Our study revealed a novel regulatory mechanism underlying root adaption to N deficiency where PuHox52 modulated a coordinated uptake of nitrate, phosphate, and iron through 'PuHox52-PuNRT1.1', 'PuHox52-PuPHL1A/PuPHL1B', and'PuHox52-PuIRT1' regulatory relationships in poplar roots.