Temperature regulation of auxin-related gene expression and its implications for plant growth.

Twenty-five years ago, a seminal paper demonstrated that warm temperatures increase auxin levels to promote hypocotyl growth in Arabidopsis thaliana. Here we highlight recent advances in auxin-mediated thermomorphogenesis and identify unanswered questions. In the warmth, PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF7 bind the YUCCA8 gene promoter and, in concert with histone modifications, enhance its expression to increase auxin synthesis in the cotyledons. Once transported to the hypocotyl, auxin promotes cell elongation. The meta-analysis of expression of auxin-related genes in seedlings exposed to temperatures ranging from cold to hot shows complex patterns of response. Changes in auxin only partially account for these responses. The expression of many SMALL AUXIN UP RNA (SAUR) genes reaches a maximum in the warmth, decreasing towards both temperature extremes in correlation with the rate of hypocotyl growth. Warm temperatures enhance primary root growth, the response requires auxin, and the hormone levels increase in the root tip but the impacts on cell division and cell expansion are not clear. A deeper understanding of auxin-mediated temperature control of plant architecture is necessary to face the challenge of global warming.

EARLY FLOWERING 3 represses the nighttime growth response to sucrose in Arabidopsis.

Plant growth depends on the supply of carbohydrates produced by photosynthesis. Exogenously applied sucrose promotes the growth of the hypocotyl in Arabidopsis thaliana seedlings grown under short days. Whether this effect of sucrose is stronger under the environmental conditions where the light input for photosynthesis is limiting remains unknown. We characterised the effects of exogenous sucrose on hypocotyl growth rates under light compared to simulated shade, during different portions of the daily cycle. The strongest effects of exogenous sucrose occurred under shade and during the night; i.e., the conditions where there is reduced or no photosynthesis. Conversely, a faster hypocotyl growth rate, predicted to enhance the demand of carbohydrates, did not associate to a stronger sucrose effect. The early flowering 3 (elf3) mutation strongly enhanced the impact of sucrose on hypocotyl growth during the night of a white-light day. This effect occurred under short, but not under long days. The addition of sucrose enhanced the fluorescence intensity of ELF3 nuclear speckles. The elf3 mutant showed increased abundance of PHYTOCHROME INTERACTING FACTOR4 (PIF4), which is a transcription factor required for a full response to sucrose. Sucrose increased PIF4 protein abundance by post-transcriptional mechanisms. Under shade, elf3 showed enhanced daytime and reduced nighttime effects of sucrose. We conclude that ELF3 modifies the responsivity to sucrose according to the time of the daily cycle and the prevailing light or shade conditions.

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

Identification and characterization of two RNA silencing suppressors encoded by ophioviruses.

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K-25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MPCPsV and 24KCPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MPCPsV and 24KCPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway.

Bioinformatic and mutational analysis of ophiovirus movement proteins, belonging to the 30K superfamily.

Ophioviridae is a family of segmented, negative-sense, single-stranded RNA plant viruses. We showed that their cell-to-cell movement protein (MP) is an isolated member of the 30K MP superfamily with a unique structural organization. All 30K MPs share a core domain that contains a nearly-invariant signature aspartate. We examined its role in the MP of Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV). Alanine substitution of this aspartate prevented plasmodesmata accumulation of MP(MiLBVV), while MP(CPsV) was not affected. The capacity of ophiovirus MPs to increase the plasmodesmata size exclusion limit and non-cell autonomous protein feature was abolished in both mutants. To investigate the role of the signature aspartate in cell-to-cell movement, we constructed a new movement-deficient Tobacco mosaic virus vector used for trans-complementation assays. We showed that both ophiovirus MP mutants lack the cell-to-cell movement capacity, confirming that this signature aspartate is essential for viral cell-to-cell movement.

Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein.

Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV), members of the Ophioviridae family, have segmented negative-sense single-stranded RNA genomes. To date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. Here we show that the 54K protein of CPsV is encoded by RNA 2 and describe its subcellular distribution. Upon transient expression in Nicotiana benthamiana epidermal cells the 54K protein, and also its 54K counterpart protein of MiLBVV, localize to plasmodesmata and enhance GFP cell-to-cell diffusion between cells. Both proteins, but not the coat proteins (CP) of the respective viruses, functionally trans-complement cell-to-cell movement-defective Potato virus X (PVX) and Tobacco mosaic virus (TMV) mutants. The 54K and 54K proteins interact with the virus-specific CP in the cytoplasm, suggesting a potential role of CP in ophiovirus movement. This is the first study characterizing the movement proteins (MP) of ophioviruses.

Citrus psorosis and Mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo.

Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction. The cytoplasmic localization of this protein was confirmed by confocal microscopy of fluorescent protein (FP)-tagged CP following its expression in either CPsV-infected and healthy Citrus sinensis plants or in Nicotiana benthamiana plants. The same localization was observed for FP-tagged CP of MiLBVV. The CPs of CPsV and MiLBBV can undergo homologous and heterologous interactions as revealed by fluorescent lifetime imaging microscopy and co-immunoprecipitation analysis. A putative leucine zipper motif that is conserved among ophiovirus CP sequences may account for these interactions.