Chlorophyll degradation is an important part of leaf senescence, but the underlying regulatory mechanisms are largely unknown. Excised leaves of an Arabidopsis thaliana NAC-LIKE, ACTIVATED BY AP3/PI (NAP) transcription factor mutant (nap) exhibited lower transcript levels of known chlorophyll degradation genes, STAY-GREEN1 (SGR1), NON-YELLOW COLORING1 (NYC1), PHEOPHYTINASE (PPH), and PHEIDE a OXYGENASE (PaO), and higher chlorophyll retention than the wild type during dark-induced senescence. Transcriptome coexpression analysis revealed that abscisic acid (ABA) metabolism/ signaling genes were disproportionately represented among those positively correlated with NAP expression. ABA levels were abnormally low in nap leaves during extended darkness. The ABA biosynthetic genes 9-CIS-EPOXYCAROTENOID DIOXYGENASE2, ABA DEFICIENT3, and ABSCISIC ALDEHYDE OXIDASE3 (AAO3) exhibited abnormally low transcript levels in dark-treated nap leaves. NAP transactivated the promoter of AAO3 in mesophyll cell protoplasts, and electrophoretic mobility shift assays showed that NAP can bind directly to a segment (2196 to 2162 relative to the ATG start codon) of the AAO3 promoter. Exogenous application of ABA increased the transcript levels of SGR1, NYC1, PPH, and PaO and suppressed the stay-green phenotype of nap leaves during extended darkness. Overexpression of AAO3 in nap leaves also suppressed the stay-green phenotype under extended darkness. Collectively, the results show that NAP promotes chlorophyll degradation by enhancing transcription of AAO3, which leads to increased levels of the senescence-inducing hormone ABA.

Nutrient management in biomass production systems serves to maximize yield and minimize production costs and environmental impact. Loss of soil nutrients with harvested biomass can be reduced by the judicious choice of genotype and harvest time. Sustainable production of switchgrass for biofuel will depend, in part, on breeding of varieties that are conservative in their use of soil nutrients to produce biomass. To aid such breeding programs, we assessed the natural variation in nutrient-use and remobilization efficiencies of 31 accessions of Panicum virgatum by measuring the concentration of 20 elements (N, and Cd) in shoots of field-grown plants harvested at two different stages of development. Significant differences between accessions were found for elemental composition at maturity and after senescence. The concentration of several elements (N, P, K, and Rb) decreased in the shoots of all accessions during senescence, although the efficiency of remobilization ranged from 20% to 61% for N, 31% to 65% for P, 25% for 84% for K, and 33% to 84% for Rb. The accessions/cultivars with the greatest nutrient-use efficiency (smallest loss of nutrient per unit biomass) were BN-14668-65, Kanlow, Caddo from the point of view of N content, and Kanlow, Cave-in-Rock, and Blackwell from the point of view of P content in senescent shoots. Finally, differences in elemental composition between upland and lowland ecotypes were also found. The information presented here will help to guide future breeding programs and nutrient management practices.

Switchgrass (Panicum virgatum L.) is a perennial C 4 grass native to North America that is being developed as a feedstock for cellulosic ethanol production. Industrial nitrogen fertilizers enhance switchgrass biomass production but add to production and environmental costs. A potential sustainable alternative source of nitrogen is biological nitrogen fixation. As a step in this direction, we studied the diversity of nitrogen-fixing bacteria (NFB) associated with native switchgrass plants from the tallgrass prairie of northern Oklahoma (United States), using a culture-independent approach. DNA sequences from the nitrogenase structural gene, nifH, revealed over 20 putative diazotrophs from the alpha-, beta-, delta-, and gammaproteobacteria and the firmicutes associated with roots and shoots of switchgrass. Alphaproteobacteria, especially rhizobia, predominated. Sequences derived from nifH RNA indicated expression of this gene in several bacteria of the alpha-, beta-, delta-, and gammaproteobacterial groups associated with roots. Prominent among these were Rhizobium and Methylobacterium species of the alphaproteobacteria, Burkholderia and Azoarcus species of the betaproteobacteria, and Desulfuromonas and Geobacter species of the deltaproteobacteria.

SummaryImproving nitrogen (N) remobilization from aboveground to underground organs during yearly shoot senescence is an important goal for sustainable production of switchgrass (Panicum virgatum) as a biofuel crop. Little is known about the genetic control of senescence and N use efficiency in perennial grasses such as switchgrass, which limits our ability to improve the process.Switchgrass aboveground organs (leaves, stems and inflorescences) and underground organs (crowns and roots) were harvested every month over a 3-yr period. Transcriptome analysis was performed to identify genes differentially expressed in various organs during development.Total N content in aboveground organs increased from spring until the end of summer, then decreased concomitant with senescence, while N content in underground organs exhibited an increase roughly matching the decrease in shoot N during fall. Hundreds of senescence-associated genes were identified in leaves and stems. Functional grouping indicated that regulation of transcription and protein degradation play important roles in shoot senescence. Coexpression networks predict important roles for five switchgrass NAC (NAM, ATAF1,2, CUC2) transcription factors (TFs) and other TF family members in orchestrating metabolism of carbohydrates, N and lipids, protein modification/degradation, and transport processes during senescence.This study establishes a molecular basis for understanding and enhancing N remobilization and conservation in switchgrass.

Two full-length cDNAs encoding NAM, ATAF, and CUC (NAC)-family transcription factors (TFs) were isolated from two different cultivars of Panicum virgatum L. (switchgrass) and named PvNAC1 and PvNAC2. Phylogenetic analysis of PvNAC1 and PvNAC2 grouped them with NAC proteins involved in senescence in annual plant species. Transcript profiling revealed that both PvNAC1 and PvNAC2 are induced during leaf senescence. Expression of a PvNAC1-green fluorescent protein (GFP) fusion in plant cells revealed a nuclear location of the protein, consistent with a role in transcriptional regulation. Expression of PvNAC1 in an Arabidopsis nap stay-green mutant suppressed its senescence defect. Expression of PvNAC1 in wild-type Arabidopsis triggered early leaf senescence and remobilization. Transcriptome analysis implicated leaf protein degradation and nitrogen recycling enzymes in NAC-dependent seed protein increase in Arabidopsis. Overexpression of pvNAC2 in switchgrass resulted in increased aboveground biomass associated with increased transcript levels of key nitrogen metabolism genes in leaves and nitrate and ammonium transporter genes in roots. The results indicate that NAC TFs play conserved roles in leaf senescence in the plant kingdom not only in annual monocots and dicots but also in perennial plants such as switchgrass. PvNAC1 and PvNAC2 hold promise for improving nutrient use efficiency in switchgrass through genetic manipulation.