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Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Orobanche cumana is a holoparasitic plant that attaches to host-plant roots and seriously reduces the yield of sunflower (Helianthus annuus L.). Effective control methods are lacking with only a few known sources of genetic resistance. In this study, a seed-soak agroinoculation (SSA) method was established, and recombinant tobacco rattle virus vectors were constructed to express RNA interference (RNAi) inducers to cause virus-induced gene silencing (VIGS) in sunflower. A host target gene HaTubulin was systemically silenced in both leaf and root tissues by the SSA-VIGS approach. Trans-species silencing of O. cumana genes were confirmed for 10 out of 11 target genes with silencing efficiency of 23.43%-92.67%. Knockdown of target OcQR1, OcCKX5, and OcWRI1 genes reduced the haustoria number, and silencing of OcEXPA6 caused further phenotypic abnormalities such as shorter tubercles and necrosis. Overexpression of OcEXPA6 caused retarded root growth in alfalfa (Medicago sativa). The results demonstrate that these genes play an important role in the processes of O. cumana parasitism. High-throughput small RNA (sRNA) sequencing and bioinformatics analyses unveiled the distinct features of target gene-derived siRNAs in O. cumana such as siRNA transitivity, strand polarity, hotspot region, and 21/22-nt siRNA predominance, the latter of which was confirmed by Northern blot experiments. The possible RNAi mechanism is also discussed by analyzing RNAi machinery genes in O. cumana. Taken together, we established an efficient host-induced gene silencing technology for both functional genetics studies and potential control of O. cumana. The ease and effectiveness of this strategy could potentially be useful for other species provided they are amenable to SSA., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.

Alfalfa (Medicago sativa) stem elongation is strongly reduced by a pea aphid (Acyrthosiphon pisum Harris) infestation. As pea aphid is a phloem feeder that does not transmit virus or toxins, assimilate withdrawal is generally considered as the main mechanism responsible for growth reduction. Using a kinematic analysis, we investigated the spatial distributions of relative elemental growth rates of control and infested alfalfa stems. The water, carbon, and nitrogen contents per unit stem length were measured along the growth zone. Deposition rates and growth-sustaining fluxes were estimated from these patterns. Severe short-term aphid infestation (200 young adults over a 24-h period) induced a strong and synchronized reduction in rates of elongation and of water and carbon deposition. Reduced nitrogen content and associated negative nitrogen deposition rates were observed in some parts of the infested stems, especially in the apex. This suggested a mobilization of nitrogen from the apical part of the growth zone, converted from a sink tissue into a source tissue by aphids. Calculation of radial growth rates suggested that aphid infestation led to a smaller reduction in radial expansion than in elongation. Together with earlier observations of long-lasting effects of a short-term infestation, this supports the hypothesis that in addition to nutrient withdrawal, a thigmomorphogenesis-like mechanism is involved in the effect of aphid infestation on stem growth.

The osmoprotectant Pro betaine is the main betaine identified in alfalfa (Medicago sativa). We have investigated the long-term responses of nodulated alfalfa plants to salt stress, with a particular interest for Pro betaine accumulation, compartmentalization, and metabolism. Exposure of 3-week-old nodulated alfalfa plants to 0.2 m NaCl for 4 weeks was followed by a 10-, 4-, and 8-fold increase in Pro betaine in shoots, roots, and nodules, respectively. Isotope-labeling studies in alfalfa shoots indicate that [14C]Pro betaine was synthesized from l-[14C]Pro. [14C]Pro betaine was efficiently catabolized through sequential demethylations via N-methylPro and Pro. Salt stress had a minor effect on Pro betaine biosynthesis, whereas it strongly reduced Pro betaine turnover. Analysis of Pro betaine and Pro compartmentalization within nodules revealed that 4 weeks of salinization of the host plants induced a strong increase in cytosol and bacteroids. The estimated Pro betaine and Pro concentrations in salt-stressed bacteroids reached 7.4 and 11.8 mm, respectively, compared to only 0.8 mm in control bacteroids. Na+ content in nodule compartments was also enhanced under salinization, leading to a concentration of 14.7 mm in bacteroids. [14C]Pro betaine and [14C]Pro were taken up by purified symbiosomes and free bacteroids. There was no indication of saturable carrier(s), and the rate of uptake was moderately enhanced by salinization. Ultrastructural analysis showed a large peribacteroid space in salt-stressed nodules, suggesting an increased turgor pressure inside the symbiosomes, which might partially be due to an elevated concentration in Pro, Pro betaine, and Na+ in this compartment.

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.

The diploid annual legume Medicago truncatula has been developed as a tractable genetic system for studying biological questions that are unique to, or well suited for study in legume species. An efficient mutagenesis protocol using ethyl-methyl sulfonate and a polymorphic ecotype with properties appropriate for use as a mapping parent are described. Isolation and characterization of three developmental mutants are described. The mtapetala mutation results in homeotic conversions of floral organ whorls 2 and 3 into sepals and carpelloid structures, respectively, similar to mutations in the apetala3/pistillata genes of Arabidopsis. The palmyra mutation primarily affects seedling shoot meristem initiation, and thus phenocopies meristem function mutations identified in Arabidopsis such as the zwille locus. The phenotype of the palmyra and mtapetala double mutant is additive, with seedling shoot meristems and floral organs indistinguishable from those of the single palmyra and mtapetala mutants, respectively. These results are consistent with a lack of genetic interaction between these loci. A third mutant, speckle, is characterized by spontaneous necrotic lesion formation on leaves, root, and stems, similar to necrosis mutants identified in other plant species. In addition to documenting the efficient mutagenesis of M. truncatula, the availability of developmental mutants that phenocopy characterized Arabidopsis mutants will provide a basis for establishing orthologous gene function between M. truncatula and Arabidopsis, once the genes responsible are cloned. Moreover, the male-sterile, female-fertile nature of the mtapetala mutant provides a convenient tool for genetic analyses in M. truncatula.

Sinorhizobium meliloti nodulation factors (NFs) elicit a number of symbiotic responses in alfalfa (Medicago sativa) roots. Using a semiquantitative nodulation assay, we have shown that chemically synthesized NFs trigger nodule formation in the same range of concentrations (down to 10(-10) M) as natural NFs. The absence of O-sulfate or O-acetate substitutions resulted in a decrease in morphogenic activity of more than 100-fold and approximately 10-fold, respectively. To address the question of the influence of the structure of the N-acyl chain, we synthesized a series of sulfated tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of different lengths and with unsaturations either conjugated to the carbonyl group (2E) or located in the middle of the chain (9Z). A nonacylated, sulfated chitin tetramer was unable to elicit nodule formation. Acylation with short (C8) chains rendered the LCO active at 10(-7) M. The optimal chain length was C16, with the C16-LCO being more than 10-fold more active than the C12- and C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO was more active than the monounsaturated LCOs. We discuss different hypotheses for the role of the acyl chain in NF perception.

MsEnod12A and MsEnod12B are two early nodulin genes from alfalfa (Medicago sativa). Differential expression of these genes was demonstrated using a reverse transcription-polymerase chain reaction approach. MsEnod12A RNA was detected only in nodules and not in other plant tissues. In contrast, MsEnod12B transcripts were found in nodules and also at low levels in roots, flowers, stems, and leaves. MsEnod12B expression was enhanced in the root early after inoculation with the microsymbiont Rhizobium meliloti and after treatment with purified Nod factors, whereas MsEnod12A induction was detected only when developing nodules were visible. In situ hybridization showed that in nodules, MsEnod12 expression occurred in the infection zone. In empty Fix- nodules the MsEnod12A transcript level was much reduced, and in spontaneous nodules it was not detectable. These data indicate that MsEnod12B expression in roots is related to the action of Nod factors, whereas MsEnod12A expression is associated with the invasion process in nodules. Therefore, alfalfa possesses different mechanisms regulating MsEnod12A and MsEnod12B expression.

The glutamine synthetase (GS) gene family of Medicago truncatula Gaertn. contains three genes related to cytosolic GS (MtGSa, MtGSb, and MtGSc), although one of these (MtGSc) appears not to be expressed. Sequence analysis suggests that the genes are more highly conserved interspecifically rather than intraspecifically: MtGSa and MtGSb are more similar to their homologs in Medicago sativa and Pisum sativum than to each other. Studies in which gene-specific probes are used show that both MtGSa and MtGSb are induced during symbiotic root nodule development, although not coordinately. MtGSa is the most highly expressed GS gene in nodules but is also expressed to lower extents in a variety of other organs. MtGSb shows higher levels of expression in roots and the photosynthetic cotyledons of seedlings than in nodules or other organs. In roots, both genes are expressed in the absence of an exogenous nitrogen source. However the addition of nitrate leads to a short-term, 2- to 3-fold increase in the abundance of both mRNAs, and the addition of ammonium leads to a 2-fold increase in MtGSb mRNA. The nitrogen supply, therefore, influences the expression of the two genes in roots, but it is clearly not the major effector of their expression. In the discussion section, the expression of the GS gene family of the model legume M. truncatula is compared to those of other leguminous plants.

When alfalfa (Medicago sativa L. cv Apica) plants grown at room temperature are transferred to 2 degrees C, the temperature at which 50% of the plants fail to survive (LT50) decreases from -6 to -14 degrees C during the first 2 weeks but then increases to -9 degrees C during the subsequent 2 weeks. However, when plants are kept for 2 weeks at 2 degrees C and then transferred to -2 degrees C for another two weeks, the LT50 declines to -16 degrees C. These changes in freezing tolerance are paralleled by changes in transcript levels of cas15 (cold acclimation-specific gene encoding a 14.5-kD protein), a cold-induced gene. Cold-activation of cas15 occurs even when protein synthesis is inhibited by more than 90%, suggesting that cold-initiated events up to and including the accumulation of cas15 transcripts depend on preexisting gene products. cas15 shows little homology to any known gene at the nucleotide or amino acid level. The deduced polypeptide (CAS15) of 14.5 kD contains four repeats of a decapeptide motif and possesses a bipartite sequence domain at the carboxy terminus with homology to the reported nuclear-targeting signal sequences. Although the relative amount of cas15 DNA as a fraction of the total genomic DNA is similar in cultivars with different degrees of freezing tolerance, its organization in the genome is different. The possible role of cas15 in the development of cold-induced freezing tolerance is discussed.